Soluble amide &amp; ester pyrazinoylguanidine sodium channel blockers

ABSTRACT

The present invention relates to sodium channel blockers. The present invention also includes a variety of methods of treatment using these inventive sodium channel blockers.

BACKGROUND OF THE INVENTION

1. Field of the Invention

The present invention relates to sodium channel blockers. The presentinvention also includes a variety of methods of treatment using theseinventive sodium channel blockers.

2. Description of the Background

The mucosal surfaces at the interface between the environment and thebody have evolved a number of “innate defense”, i.e., protectivemechanisms. A principal form of such innate defense is to cleanse thesesurfaces with liquid. Typically, the quantity of the liquid layer on amucosal surface reflects the balance between epithelial liquidsecretion, often reflecting anion (Cl⁻ and/or HCO₃ ⁻) secretion coupledwith water (and a cation counter-ion), and epithelial liquid absorption,often reflecting Na⁺ absorption, coupled with water and counter anion(Cl⁻ and/or HCO₃ ⁻). Many diseases of mucosal surfaces are caused by toolittle protective liquid on those mucosal surfaces created by animbalance between secretion (too little) and absorption (relatively toomuch). The defective salt transport processes that characterize thesemucosal dysfunctions reside in the epithelial layer of the mucosalsurface.

One approach to replenish the protective liquid layer on mucosalsurfaces is to “re-balance” the system by blocking Na⁺ channel andliquid absorption. The epithelial protein that mediates therate-limiting step of Na⁺ and liquid absorption is the epithelial Na⁺channel (ENaC). ENaC is positioned on the apical surface of theepithelium, i.e. the mucosal surface-environmental interface. Therefore,to inhibit ENaC mediated Na⁺ and liquid absorption, an ENaC blocker ofthe amiloride class (which blocks from the extracellular domain of ENaC)must be delivered to the mucosal surface and, importantly, be maintainedat this site, to achieve therapeutic utility. The present inventiondescribes diseases characterized by too little liquid on mucosalsurfaces and “topical” sodium channel blockers designed to exhibit theincreased potency, reduced mucosal absorption, and slow dissociation(“unbinding” or detachment) from ENaC required for therapy of thesediseases.

Chronic bronchitis (CB), including the most common lethal genetic formof chronic bronchitis, cystic fibrosis (CF), are diseases that reflectthe body's failure to clear mucus normally from the lungs, whichultimately produces chronic airways infection. In the normal lung, theprimary defense against chronic intrapulmonary airways infection(chronic bronchitis) is mediated by the continuous clearance of mucusfrom bronchial airway surfaces. This function in health effectivelyremoves from the lung potentially noxious toxins and pathogens. Recentdata indicate that the initiating problem, i.e., the “basic defect,” inboth CB and CF is the failure to clear mucus from airway surfaces. Thefailure to clear mucus reflects an imbalance between the amount ofliquid and mucin on airway surfaces. This “airway surface liquid” (ASL)is primarily composed of salt and water in proportions similar to plasma(i.e., isotonic). Mucin macromolecules organize into a well defined“mucus layer” which normally traps inhaled bacteria and is transportedout of the lung via the actions of cilia which beat in a watery, lowviscosity solution termed the “periciliary liquid” (PCL). In the diseasestate, there is an imbalance in the quantities of mucus as ASL on airwaysurfaces. This results in a relative reduction in ASL which leads tomucus concentration, reduction in the lubricant activity of the PCL, anda failure to clear mucus via ciliary activity to the mouth. Thereduction in mechanical clearance of mucus from the lung leads tochronic bacterial colonization of mucus adherent to airway surfaces. Itis the chronic retention of bacteria, the failure of local antimicrobialsubstances to kill mucus-entrapped bacteria on a chronic basis, and theconsequent chronic inflammatory responses of the body to this type ofsurface infection, that lead to the syndromes of CB and CF.

The current afflicted population in the U.S. is 12,000,000 patients withthe acquired (primarily from cigarette smoke exposure) form of chronicbronchitis and approximately 30,000 patients with the genetic form,cystic fibrosis. Approximately equal numbers of both populations arepresent in Europe. In Asia, there is little CF but the incidence of CBis high and, like the rest of the world, is increasing.

There is currently a large, unmet medical need for products thatspecifically treat CB and CF at the level of the basic defect that causethese diseases. The current therapies for chronic bronchitis and cysticfibrosis focus on treating the symptoms and/or the late effects of thesediseases. Thus, for chronic bronchitis, β-agonists, inhaled steroids,anti-cholinergic agents, and oral theophyllines and phosphodiesteraseinhibitors are all in development. However, none of these drugs treateffectively the fundamental problem of the failure to clear mucus fromthe lung. Similarly, in cystic fibrosis, the same spectrum ofpharmacologic agents is used. These strategies have been complemented bymore recent strategies designed to clear the CF lung of the DNA(“Pulmozyme”; Genentech) that has been deposited in the lung byneutrophils that have futilely attempted to kill the bacteria that growin adherent mucus masses and through the use of inhaled antibiotics(“TOBI”) designed to augment the lungs' own killing mechanisms to ridthe adherent mucus plaques of bacteria. A general principle of the bodyis that if the initiating lesion is not treated, in this case mucusretention/obstruction, bacterial infections became chronic andincreasingly refractory to antimicrobial therapy. Thus, a major unmettherapeutic need for both CB and CF lung diseases is an effective meansof re-hydrating airway mucus (i.e., restoring/expanding the volume ofthe ASL) and promoting its clearance, with bacteria, from the lung.

R. C. Boucher, in U.S. Pat. No. 6,264,975, describes the use ofpyrazinoylguanidine sodium channel blockers for hydrating mucosalsurfaces. These compounds, typified by the well-known diureticsamiloride, benzamil, and phenamil, are effective. However, thesecompounds suffer from the significant disadvantage that they are (1)relatively impotent, which is important because the mass of drug thatcan be inhaled by the lung is limited; (2) rapidly absorbed, whichlimits the half-life of the drug on the mucosal surface; and (3) arefreely dissociable from ENaC. The sum of these disadvantages embodied inthese well known diurectics produces compounds with insufficient potencyand/or effective half-life on mucosal surfaces to have therapeuticbenefit for hydrating mucosal surfaces.

Clearly, what is needed are drugs that are more effective at restoringthe clearance of mucus from the lungs of patients with CB/CF. The valueof these new therapies will be reflected in improvements in the qualityand duration of life for both the CF and the CB populations.

Other mucosal surfaces in and on the body exhibit subtle differences inthe normal physiology of the protective surface liquids on theirsurfaces but the pathophysiology of disease reflects a common theme,i.e., too little protective surface liquid. For example, in xerostomia(dry mouth) the oral cavity is depleted of liquid due to a failure ofthe parotid sublingual and submandibular glands to secrete liquiddespite continued Na⁺ (ENaC) transport mediated liquid absorption fromthe oral cavity. Similarly, keratoconjunctivitis sira (dry eye) iscaused by failure of lacrimal glands to secrete liquid in the face ofcontinued Na⁺ dependent liquid absorption on conjunctional surfaces. Inrhinosinusitis, there is an imbalance, as in CB, between mucin secretionand relative ASL depletion. Finally, in the gastrointestinal tract,failure to secrete Cl—(and liquid) in the proximal small intestine,combined with increased Na⁺ (and liquid) absorption in the terminalileum leads to the distal intestinal obstruction syndrome (DIOS). Inolder patients excessive Na⁺ (and volume) absorption in the descendingcolon produces constipation and diverticulitis.

Fifty million Americans and hundreds of millions of others around theworld suffer from high blood pressure and the subsequent sequale leadingto congestive heart failure and increasing mortality. It is the WesternWorld's leading killer and there is a need there for new medicines totreat these diseases. Thus, in addition, some of the novel sodiumchannel blockers of this invention can be designed to target the kidneyand as such they may be used as diuretics for the treatment ofhypertension, congestive heart failure (CHF) and other cardiovasculardiseases. These new agents may be used alone or in combination withbeta-blockers, ACE inhibitors, HMGCoA reductase inhibitors, calciumchannel blockers and other cardiovascular agents.

SUMMARY OF THE INVENTION

It is an object of the present invention to provide compounds that aremore potent and/or absorbed less rapidly from mucosal surfaces, and/orare less reversible as compared to known compounds.

It is another aspect of the present invention to provide compounds offormula (I) that are more potent and/or absorbed less rapidly and/orexhibit less reversibility, as compared to compounds such as amilorde,benzamil, and phenamil. Therefore, the compounds of formula (I) willgive a prolonged pharmacodynamic half-life on mucosal surfaces ascompared to known compounds.

It is another aspect of the present invention to provide compounds offormula (I) that are more soluble in aqueous solutions, especially in0.12-0.9% saline, so that they can be conveniently administered tomucosal surfaces of a patient by suitable means such as a nebulizer,spay, mist or droplets. Therefore, the compounds of formula (I) are moresoluble in aqueous solutions as compared to known compounds lacking theadditional proanatable nitrogen(s) contained in compounds of Formula (I)

It is another object of the present invention to provide compounds offormula (I) which are (1) absorbed less rapidly from mucosal surfaces,especially airway surfaces, as compared to known compounds and; (2) whenabsorbed from musosal surfaces after administration to the mucosalsurfaces, are converted in vivo into metabolic derivitives thereof whichhave reduced efficacy in blocking sodium channels as compared to theadministered parent compound.

It is another object of the present invention to provide compounds offormula (1) that are more potent and/or absorbed less rapidly and/orexhibit less reversibility, as compared to compounds such as amiloride,benzamil, and phenamil. Therefore, the compounds of formula (I) willgive a prolonged pharmacodynamic half-life on mucosal surfaces ascompared to previous compounds.

It is another object of the present invention to provide compounds offormula (I) that target the kidney for use in the treatment ofcardiovascular disease.

It is another object of the present invention to provide methods oftreatment which take advantage of the properties described above.

The objects of the present invention may be accomplished with a class ofpyrazinoylguanidine compounds represented by formula (I):

where X is hydrogen, halogen, trifluoromethyl, lower alkyl,unsubstituted or substituted phenyl, lower alkyl-thio, phenyl-loweralkyl-thio, lower alkyl-sulfonyl, or phenyl-lower alkyl-sulfonyl; Y ishydrogen, hydroxyl, mercapto, lower alkoxy, lower alkyl-thio, halogen,lower alkyl, unsubstituted or substituted mononuclear aryl, or —N(R²)₂;R¹ is hydrogen or lower alkyl; each R² is, independently, —R⁷,—(CH₂)_(m)—OR⁸, —(CH₂)_(m)—NR⁷R¹⁰, —(CH₂)_(n)(CHOR⁸)(CHOR⁸)_(n)—CH₂OR⁸,—(CH₂CH₂O)_(m)—R⁸, —(CH₂CH₂O)_(m)—CH₂CH₂NR⁷R₁₀, —(CH₂)_(n)—C(═O)NR⁷R¹⁰,—(CH₂)_(n)-Z_(g)-R⁷,—(CH₂)_(m)—NR¹⁰—CH₂(CHOR⁸)(CHOR⁸)_(n)—CH₂OR⁸,—(CH₂)_(n)—CO₂R⁷, or

wherein when two —CH₂OR⁸ groups are located 1,2- or 1,3- with respect toeach other the R⁸ groups may be joined to form a cyclic mono- ordi-substituted 1,3-dioxane or 1,3-dioxolane; R³ and R⁴ are each,independently, hydrogen, a group represented by formula (A), loweralkyl, hydroxy lower alkyl, phenyl, phenyl-lower alkyl,(halophenyl)-lower alkyl, lower-(alkylphenylalkyl), lower(alkoxyphenyl)-lower alkyl, naphthyl-lower alkyl, or pyridyl-loweralkyl, with the proviso that at least one of R³ and R⁴ is a grouprepresented by formula (A):

wherein

each R^(L) is, independently, —R⁷, —(CH₂)_(n)—OR⁸, —O—(CH₂)_(m)—OR⁸,—(CH₂)_(n)—NR⁷R¹⁰, —O—(CH₂)_(m)—NR⁷R¹⁰,—(CH₂)_(n)(CHOR⁸)(CHOR⁸)_(n)—CH₂OR⁸,—O—(CH₂)_(m)(CHOR⁸)(CHOR⁸)_(n)—CH₂OR⁸, —(CH₂CH₂O)_(m)—R⁸,—O—(CH₂CH₂O)_(m)—R⁸, —(CH₂CH₂O)_(m)—CH₂CH₂NR⁷R¹⁰,—O—(CH₂CH₂O)_(m)—CH₂CH₂NR⁷R¹⁰, —(CH₂)_(n)—C(═O)NR⁷R¹⁰,—O—(CH₂)_(m)—C(═O)NR⁷R¹⁰, —(CH₂)_(n)-(Z)_(g)-R⁷, —O—(CH₂)_(m)(Z)_(g)-R⁷,—(CH₂)_(n)—NR¹⁰—CH₂(CHOR⁸)(CHOR⁸)_(n)—CH₂OR⁸,—O—(CH₂)_(m)—NR¹⁰—CH₂(CHOR⁸)(CHOR⁸)_(n)—CH₂OR⁸, —(CH₂)_(n)—CO₂R⁷,—O—(CH₂)_(m)—CO₂R⁷, —OSO₃H, —O-glucuronide, —O-glucose,

wherein when two —CH₂OR⁸ groups are located 1,2- or 1,3- with respect toeach other the R⁸ groups may be joined to form a cyclic mono- ordi-substituted 1,3-dioxane or 1,3-dioxolane;

each o is, independently, an integer from 0 to 10;

each p is an integer from 0 to 10;

with the proviso that the sum of o and p in each contiguous chain isfrom 1 to 10;

each x is, independently, O, NR¹⁰, C(═O), CHOH, C(αN—R¹⁰), CHNR⁷R¹⁰, orrepresents a single bond;

each R⁵ is independently, —(CH₂)_(n)—CO₂R³, Het-(CH₂)_(m)—CO₂R¹³,—(CH₂)_(n)-Z_(g)-CO₂R¹³, Het-(CH₂)_(m)-Z_(g)-CO₂R¹³,—(CH₂)_(n)—NR¹⁰—(CH₂)_(m)(CHOR⁸)_(n)—CO₂R¹³,Het-(CH₂)_(m)—NR¹⁰—(CH₂)_(m)(CHOR⁸)_(n)—CO₂R¹³,—(CH₂)_(n)—(CHOR⁸)_(m)—CO₂R¹³, Het-(CH₂)_(m)—(CHOR⁸)_(m)—CO₂R¹³,—(CH₂)_(n)—(CHOR⁸)_(m)Z_(g)-CO₂R¹³,Het-(CH₂)_(n)—(CHOR⁸)_(m)-Z_(g)-CO₂R¹³,—(CH₂)_(n)-Z_(g)-(CH₂)_(m)—CO₂R¹³, —(CH₂)_(n)-Z_(g)-(CH₂)_(m)—CO₂R¹³,—(CH₂)_(n)-Z_(g)(CHOR⁸)_(m)-Z_(g)-CO₂R¹³,Het-(CH₂)_(n)-Z_(g)-(CHOR⁸)_(m)-Z_(g)-CO₂R¹³,—(CH₂)_(n)—CONH—C(═NR¹³)—NR¹³R¹³, Het-(CH₂)_(n)—CO—NH—C(═NR¹³)—NR¹³R¹³,—(CH₂)_(n)-Z_(g)-CONH—C(═NR¹³)—NR¹³R¹³,Het-(CH₂)_(n)-Z_(g)-CONH—C(—NR¹³)—NR¹³R¹³,—(CH₂)_(n)—NR¹⁰—(CH₂)_(m)(CHOR⁸)_(n)—CONH—C(═NR¹³)—NR13R¹³,Het-(CH₂)_(n)—NR¹⁰—(CH₂)_(m)(CHOR⁸)_(n)—CONH—C(═NR¹³)—NR¹³R¹³,—(CH₂)_(n)—(CHOR⁸)_(m)—CONH—C(═NR¹³)—NR¹³R¹³,Het-(CH₂)_(n)—(CHOR⁸)_(m)—CONH—C(═NR¹³)—NR¹³R¹³,—(CH₂)_(n)—(CHOR⁸)_(m)-Z_(g)-CONH—C(═NR¹³)—NR¹³R¹³,Het-(CH₂)_(n)—(CHOR⁸)_(m)-Z_(g)-CONH—C(═NR¹³)—NR¹³R¹³,—(CH₂)_(n)-Z_(g)-(CH₂)_(m)CONH—C(═NR¹³)—NR¹³R¹³,Het-(CH₂)_(n)-Z_(g)-(CH₂)_(m)CONH—C(═NR ¹³)—NR¹³R¹³,—(CH₂)_(n)-Z_(g)-(CHOR⁸)_(m)-Z_(g)-CONH—C(═NR¹³)—NR¹³R¹³,Het-(CH₂)_(n)-Z_(g)-(CHOR⁸)_(m)-Z_(g)-CONH—C(═NR¹³)—NR¹³R¹³,—(CH₂)_(n)—CONR⁷—CONR¹³R¹³, Het-(CH₂)_(n)—CONR⁷—CONR¹³R¹³,—(CH₂)_(n)-_(Z)g-CONR⁷—CONR¹³R¹³, —(CH₂)_(n)-Z_(g)-CONR⁷—CONR¹³R¹³,—(CH₂)_(n)—NR¹⁰—(CH₂)_(m)(CHOR⁸)_(n)—CONR⁷—CONR¹³R¹³,Het-(CH₂)_(n)—NR¹⁰—(CH₂)_(m)(CHOR⁸)_(n)-CONR⁷—CONR¹³R¹³,—(CH₂)_(n)—(CHOR⁸)_(m)—CONR⁷—CONR¹³R¹³,Het-(CH₂)_(n)—(CHOR⁸)_(m)—CONR⁷—CONR¹³R¹³,—(CH₂)_(n)—(CHOR⁸)_(m)-Z_(g)-CONR⁷—CONR¹³R¹³,Het-(CH₂)_(n)—(CHOR⁸)_(n)-Z_(g)-CNR⁷—CONR¹³R¹³,—(CH₂)_(n)-Z_(g)-(CH₂)_(m)CONR⁷—CONR¹³R¹³,Het-(CH₂)_(n)-Z_(g)-(CH₂)_(m)CONR⁷-CONR¹³R¹³,—(CH₂)_(n)-Z_(g)(CHOR⁸)_(m)-Z_(g)-CONR⁷—CONR¹³R¹³,Het-(CH₂)_(n)-Z_(g)(CHOR⁸)_(m)-Z_(g)-CONR⁷—CONR¹³R¹³,(CH₂)_(n)-CONR⁷SO₂NR¹³R¹³, Het-(CH₂)_(m)—CONR⁷SO₂NR¹³R¹³,—(CH₂)_(n)-Z_(g)(CONR⁷SO₂NR¹³R¹³, Het-(CH₂)_(m)-Z_(g)-CONR⁷SO₂NR¹³R¹³,—(CH₂)_(n)—NR¹⁰—(CH₂)_(m)(CHOR⁸)_(n)—CONR⁷SO₂NR¹³R¹³,Het-(CH₂)_(m)-NR¹⁰—(CH₂)_(m)(CHOR⁸)_(n)—CONR⁷SO₂NR¹³R¹³,—(CH₂)_(n)—(CHOR⁸)_(m)—CONR⁷SO₂NR¹³R¹³,Het-(CH₂)_(m)—(CHOR⁸)_(m)—CONR⁷SO₂NR¹³R¹³,—(CH₂)_(n)—(CHOR⁸)_(m)-Z_(g)-CONR⁷SO₂NR¹³R¹³,Het-(CH₂)_(n)—(CHOR⁸)_(m)-Z_(g)-CONR⁷SO₂NR¹³R¹³,—(CH₂)_(n)-Z_(g)-(CH₂)_(m)CONR⁷SO₂NR¹³R¹³,Het-(CH₂)_(n)-Z_(g)-(CH₂)_(m)CONR⁷SO₂NR¹³R¹³,—(CH₂)_(n)-Z_(g)-(CHOR⁸)_(m)-Z_(g)-CONR⁷SO₂NR¹³R¹³,Het-(CH₂)_(n)-Z_(g)-(CHOR⁸)_(m)-Z_(g)-CONR⁷SO₂NR¹³R¹³,—(CH₂)_(n)—SO₂NR¹³R¹³, Het-(CH₂)_(m)—SO₂NR¹³R¹³,—(CH₂)_(n)-Z_(g)-SO₂NR¹³R¹³, Het-(CH₂)_(m)-Z_(g)-SO₂NR¹³R¹³,—(CH₂)_(n)NR¹⁰—(CH₂)_(m)(CHOR⁸)_(n)—SO₂NR¹³R¹³,Het-(CH₂)_(m)—NR¹⁰—(CH₂)_(m)(CHOR⁸)_(n)—SO₂NR¹³R¹³,—(CH₂)_(n)—(CHOR⁸)_(m)—SO₂NR¹³R¹³, Het-(CH₂)_(m)—(CHOR⁸)_(m)—SO₂NR¹³R¹³,—(CH₂)_(n)—(CHOR⁸)_(m)-Z_(g)-SO₂NR¹³R¹³,Het-(CH₂)_(n)—(CHOR⁸)_(m)-Z_(g)-SO₂NR¹³R¹³,—(CH₂)_(n)-Z_(g)-(CH₂)_(m)SO₂NR¹³R¹³,Het-(CH₂)_(n)-Z_(g)-(CH₂)_(m)SO₂NR¹³R¹³,—(CH₂)_(n)-Z_(g)-(CHOR⁸)_(m)-Z_(g)-SO₂NR¹³R¹³,Het-(CH₂)_(n)-Z_(g)-(CHOR⁸)_(m)-Z_(g)-SO₂NR¹³R¹³, —(CH₂)_(n)—CONR¹³R¹³,Het-(CH₂)_(m)-CONR¹³R¹³, —(CH₂)_(n)-Z_(g)-CONR¹³R¹³,Het-(CH₂)_(m)-Z_(g)-CONR¹³R¹³,—(CH₂)_(n)—NR¹⁰—(CH₂)_(m)(CHOR⁸)_(n)—CONR¹³R¹³,Het-(CH₂)_(m)—NR¹⁰—(CH₂)_(m)(CHOR⁸)_(n)—CONR¹³R¹³,—(CH₂)_(n)—(CHOR⁸)_(m)—CONR¹³R¹³, Het-(CH₂)_(m)—(CHOR⁸)_(m)-CONR¹³R¹³,—(CH₂)_(n)—(CHOR⁸)_(m)-Z_(g)-CONR¹³R¹³,Het-(CH₂)_(n)—(CHOR⁸)_(m)-Z_(g)-CONR¹³R¹³,—(CH₂)_(n)-Z_(g)-(CH₂)_(m)CONR¹³R¹³,Het-(CH₂)_(n)-Z_(g)-(CH₂)_(m)CONR¹³R¹³,—(CH₂)_(n)-Z_(g)-(CHOR⁸)_(m)-Z_(g)-CONR¹³R¹³,Het-(CH₂)_(n)-Z_(g)-(CHOR⁸)_(m)-Z_(g)-CONR¹³R¹³, —(CH₂)_(n)—CONR⁷COR¹³,Het-(CH₂)_(m)—CONR⁷COR¹³, —(CH₂)_(n)-Z_(g)-CONR⁷COR¹³,Het-(CH₂)_(m)-Z_(g)-CONR⁷COR¹³,—(CH₂)_(n)—NR¹⁰—(CH₂)_(m)(CHOR⁸)_(n)—CONR⁷COR¹³,Het-(CH₂)_(m)-NR¹⁰—(CH₂)_(m)(CHOR⁸)_(n)—CONR⁷COR¹³,—(CH₂)_(n)—(CHOR⁸)_(m)—CONR⁷COR¹³, Het-(CH₂)_(m)—(CHOR⁸)_(m)—CONR⁷COR¹³,—(CH₂)_(n)—(CHOR⁸)_(m)-Z_(g)-CONR⁷COR¹³,Het-(CH₂)_(n)—(CHOR⁸)_(m)-Z_(g)-CONR⁷COR¹³,—(CH₂)_(n)-Z_(g)-(CH₂)_(m)CONR⁷COR¹³, —(CH₂)_(n)-Z_(g),(CH₂)_(m)CONR⁷COR³, Het-(CH₂)_(n)-Z_(g)-(CHOR⁸)_(m)-Z_(g)-CONR⁷COR¹³,—(CH₂)_(n)—CONR⁷CO₂R¹³, —(CH₂)_(n)-Z_(g)-CONR⁷CO₂R¹³,Het-(CH₂)_(m)-Z_(g)-CONR⁷CO₂R¹³,—(CH₂)_(n)—NR¹⁰—(CH₂)_(m)(CHOR⁸)_(n)—CONR⁷CO₂R¹³,Het-(CH₂)_(m)—NR¹⁰—(CH₂)_(m)(CHOR⁸)_(n)—CO NR⁷CO₂R¹³,—(CH₂)_(n)—(CHOR⁸)_(m)—CONR⁷CO₂R¹³,Het-(CH₂)_(m)—(CHOR⁸)_(m)—CONR⁷CO₂R¹³,—(CH₂)_(n)—(CHOR⁸)_(m)-Z_(g)-CONR⁷CO₂R¹³,Het-(CH₂)_(n)—(CHOR⁸)_(m)-Z_(g)-CONR⁷CO₂R¹³,—(CH₂)_(n)-Z_(g)-(CH₂)_(m)CONR⁷CO₂R¹³,Het-(CH₂)_(n)-Z_(g)-(CH₂)_(m)CONR⁷CO₂R¹³,—(CH₂)_(n)-Z_(g)-(CHOR⁸)_(m)-Z_(g)-CONR⁷CO₂R¹³,Het-(CH₂)_(n)-Z_(g)-(CHOR⁸)_(m)-Z_(g)-CONR⁷CO₂R¹³,—(CH₂)_(n)—NH—C(═NR¹³)—NR¹³R¹³, Het-(CH₂)_(m)—NH—C(═NR¹³)—NR¹³R¹³,—(CH₂)_(n)-Z_(g)-NH—C(═NR¹³)—NR¹³R¹³,Het-(CH₂)_(m)-Z_(g)-NH—C(═NR¹³)—NR¹³R¹³,—(CH₂)_(n)—NR¹⁰—(CH₂)_(m)(CHOR⁸)_(n)—NH—C(═NR¹³)—NR¹³R¹³,Het-(CH₂)_(m)-NR¹⁰—(CH₂)_(m)(CHOR⁸)_(n)—NH—C(═NR¹³)—NR¹³R¹³,—(CH₂)_(n)—(CHOR⁸)_(m)—NH—C(═NR¹³)—NR¹³R¹³,Het-(CH₂)_(m)—(CHOR⁸)_(m)—NH—C(═NR¹³)—NR¹³R¹³,—(CH₂)_(n)—(CHOR⁸)_(m)-Z_(g)-NH—C(═NR¹³)—NR¹³R¹³,Het-(CH₂)_(n)—(CHOR⁸)_(m)-Z_(g)-NH—C(═NR¹³)—NR¹³R¹³—(CH₂)_(n)-Z_(g)-(CH₂)_(m)NH—C(═NR¹³)—NR¹³R¹³,Het-(CH₂)_(n)-Z_(g)-(CH₂)_(m)NH—C(═NR¹³)—NR¹³R¹³,—(CH₂)_(n)-Z_(g)-(CHOR⁸)_(m)-Z_(g)-NH—C(═NR¹³)—NR¹³R¹³,Het-(CH₂)_(n)-Z_(g)-(CHOR⁸)_(m)-Z_(g)-NH—C(═NR¹³)—NR¹³R¹³,—(CH₂)_(n)—C(═NR¹³)—NR¹³R¹³, Het-(CH₂)_(m)—C(═NH)—NR¹³R¹³,—(CH₂)_(n)-Z_(g)-C(═NH)—NR¹³R¹³, Het-(CH₂)_(m)-Z_(g)-C(═NH)—NR¹³R¹³,—(CH₂)_(n)—NR¹⁰—(CH₂)_(m)(CHOR⁸)_(n)—C(═NR¹³)—NR¹³R¹³,Het-(CH₂)_(m)-NR¹³R¹³, Het-(CH₂)_(m)(CHOR⁸)_(n)—C(═NR¹³)—NR¹³R¹³,—(CH₂)_(n)—(CHOR⁸)_(n)—C(═NR¹³)—NR¹³R¹³,Het-(CH₂)_(m)—(CHOR⁸)_(m)—C(═NR¹³)—NR¹³R¹³,—(CH₂)_(n)—(CHOR⁸)_(m)-Z_(g)-C(═NR¹³)—NR¹³R¹³,Het-(CH₂)_(n)—(CHOR⁸)_(m)Z_(g)-C(NR¹³)—NR¹³R¹³,—(CH₂)_(n)-Z_(g)-(CH₂)_(m)—C(═NHC(═NR¹³)—NR¹³R¹³,Het-(CH₂)_(n)-Z_(g)-(CH₂)_(m)—C(═NR¹³)—NR¹³R¹³,—(CH₂)_(n)-Z_(g)-(CHOR⁸)_(m)-Z_(g)-C(═NR¹³)—NR¹³R¹³,Het-(CH₂)_(n)-Z_(g)-(CHOR⁸)_(m)-Z_(g)-C(—NR¹³)—NR¹³R¹³;

wherein when two —CH₂OR⁸ groups are located 1,2- or 1,3- with respect toeach other the R⁸ groups may be joined to form a cyclic mono- ordi-substituted 1,3-dioxane or 1,3-dioxolane;

each R⁶ is, independently, —R⁵, —R⁷, —OR⁸, —N(R⁷)₂, —(CH₂)_(m)—OR⁸,—O—(CH₂)_(m)—OR⁸, —(CH₂)_(n)—NR⁷R¹⁰, —O—(CH₂)_(m)—NR⁷R¹⁰,—(CH₂)_(n)(CHOR⁸)(CHOR⁸)_(n)—CH₂OR⁸,—O—(CH₂)_(m)(CHOR⁸)(CHOR⁸)_(n)—CH₂OR⁸, —(CH₂CH₂O)_(m)—R⁸,—O—(CH₂CH₂O)_(m)—R⁸, —(CH₂CH₂O)_(m)—CH₂CH₂NR⁷R¹⁰,—O—(CH₂CH₂O)_(m)—CH₂CH₂NR⁷R¹⁰, —(CH₂)_(n)—C(═O)NR⁷R¹⁰,—O—(CH₂)_(m)—C(═O)NR⁷R¹⁰,—(CH₂)_(n)-(Z)_(g)-R⁷,—O—(CH₂)_(m)-(Z)_(g)-R⁷,—(CH₂)_(n)—NR¹⁰—CH₂(CHOR⁸)(CHOR⁸)_(n)—CH₂OR⁸,—O—(CH₂)_(m)—NR¹⁰—CH₂(CHOR⁸)(CHOR⁸)_(n)—CH₂OR⁸, —(CH₂)_(n)—CO₂R⁷,—O—(CH₂)_(m)—CO₂R⁷, —OSO₃H, —O-glucuronide, —O-glucose,

wherein when two R⁶ are —OR¹¹ and are located adjacent to each other ona phenyl ring, the alkyl moieties of the two R⁶ may be bonded togetherto form a methylenedioxy group, and

wherein when two —CH₂OR⁸ groups are located 1,2- or 1,3- with respect toeach other the R⁸ groups may be joined to form a cyclic mono- ordi-substituted 1,3-dioxane or 1,3-dioxolane;

each R⁷ is, independently, hydrogen lower alkyl, phenyl, substitutedphenyl or —CH₂(CHOR)⁸ _(m)—R¹⁰;

each R⁸ is, independently, hydrogen, lower alkyl, —C(═O)—R¹¹,glucuronide, 2-tetrahydropyranyl, or

each R⁹ is, independently, —CO₂R⁷, —CON(R⁷)₂, —SO₂CH₃, or —C(═O)R⁷;

each R¹⁰ is, independently, —H, —SO₂CH₃, —CO₂R⁷, —C(═O)NR⁷R⁹, —C(═O)R⁷,or —(CH₂)_(m)—(CHOH)_(n)—CH₂OH;

each Z is, independently, CHOH, C(═O), —(CH₂)_(n)—,CHNR⁷R¹⁰, C═NR¹⁰, orNR¹⁰;

each R¹¹ is, independently, lower alkyl;

each R¹² is independently, —SO₂CH₃, —CO₂R⁷, —C(═O)NR⁷R⁹, —C(═O)R⁷, or—CH₂—(CHOH)_(n)—CH₂OH;

each R¹³ is, independently, hydrogen, R⁷, R¹⁰, —(CH₂)_(m)—NR⁷R¹⁰,

with the proviso that at least one R¹³ must be a group other thanhydrogen, R⁷, or R¹⁰;

with the further proviso that NR¹³R¹³ can be joined on itself to form aring comprising one of the following:

each Het is independently, —NR⁷,—NR¹⁰, —S—, —SO—, or —SO₂—; —O—,—SO₂NH—, —NHSO₂—, —NR⁷CO—, or —CONR⁷—;

each g is, independently, an integer from 1 to 6;

each m is, independently, an integer from 1 to 7;

each n is, independently, an integer from 0 to 7;

each Q is, independently, C—R⁵, C—R⁶, or a nitrogen atom, wherein atmost three Q in a ring are nitrogen atoms;

each V is, independently,

with the proviso that when V is attached directly to a nitrogen atom,then V can also be, independently, R⁷, R¹⁰, or (R¹¹)₂;

wherein for any of the above compounds when two —CH₂OR⁸ groups arelocated 1,2- or 1,3- with respect to each other the R⁸ groups may bejoined to form a cyclic mono- or di-substituted 1,3-dioxane or1,3-dioxolane;

wherein any of the above compounds can be a pharmaceutically acceptablesalt thereof, and wherein the above compounds are inclusive of allenantiomers, diastereomers, and racemic mixtures thereof.

In a preferred embodiment, each —(CH₂)_(n)-Z_(g)-C(═NH)—NR¹³R¹³ fallswithin the scope of the structures described above and is,independently, —(CH₂)_(n)—CHNH₂(C═N)—NR¹³R¹³.

In another preferred embodiment, each Het-(CH₂)_(m)—NH—C(═NH)—NR¹³R¹³falls within the scope of the structures described above and is,independently, —(CH₂)_(n)—NH—C(═NH)NHR¹³.

In another preferred embodiment, each—(CH₂)_(n)-Z_(g)-(CHOR⁸)_(m)-Z_(g)-CONR¹³R¹³ falls within the scope ofthe structures described above and is, independently,—(CH₂)_(n)—CONHCH₂(CHOH)_(m)—CONHR¹³.

In another preferred embodiment, eachHet-(CH₂)_(n)-Z_(g)-(CHOR⁸)_(m)-Z_(g)-CONR¹³R¹³ falls within the scopeof the structures described above and is, independently,—NH—C(═O)—CH₂—(CHOH)_(n)CH₂CONR¹³R¹³.

In another a preferred embodiment, eachHet-(CH₂)_(m)-Z_(g)-C(═NH)—NR¹³R¹³ falls within the scope of thestructures described above and is, independently,—O—(CH₂)_(m)—NH—C(═NH)—N(R¹³)₂.

In another a preferred embodiment, each Het-(CH₂)_(m)-Z_(g)-CONR¹³R¹³falls within the scope of the structures described above and is,independently, —O—(CH₂)_(m)—CHNH₂—CO₂NR¹³R¹³.

In another preferred embodiment, each R⁵ falls within the scope of thestructures described above and is, independently:

—O—CH₂CHOHCH₂CONR¹³R¹³

—OCH2CHOHCH₂CO₂R¹³OCH₂CH₂CONR¹³R¹³

—OCH₂CH₂NHCOR¹³

—CH₂CH₂CONR¹³R¹³

—OCH₂CH₂CONR¹³R¹³O—(CH₂)_(m)—CO₂R¹³

—(CH₂)_(m)—CO₂R¹³

—OCH₂CH₂CO₂R¹³

—OCH₂CO₂R¹³

—O—(CH₂)_(m)—NH—C(═NH)—NR¹³)₂

—(CH₂)_(n)—NH—C(═NH)—N(R¹³)₂

—NHCH₂(CHOH)₂—CCONR¹³R¹³

—OCH₂CO₂R¹³

—NHSO₂(CH2)₂CONR¹³R¹³

—(CH₂)_(m)—NH—C(═O)—OR¹³

—O—(CH₂)_(m)—NH—C(═O)—OR¹³

—(CH₂)_(n)—NH—C(═O)—R¹³

—O—(CH₂)_(m)—NH—C(═O)—R¹³

—O—CH₂C(═ONR¹³R¹³

—CH₂NCO₂R¹³

—NHCO₂R¹³

—OCH₂CH₂CH₂CH₂CONR¹³R¹³

—SO₂CH₂CH₂CONR¹³R¹³

—OCH₂CH₂CHOHCH₂CONR¹³R¹³

—OCH₂CH₂NHCO₂R¹³

—NH—C(═NH2)—NR¹³R¹³

—OCH₂-(α-CHOH)₂—CONR¹³R¹³

—OCH₂CHOHCH₂CONHR¹³

—(CH₂)_(m)—CHOH—CH₂—NHCO₂R¹³

—O—(CH₂)_(m)—CHOH—CH₂—CO₂R¹³

—(CH₂)_(m)—NHC(O)OR¹³

—O—(CH₂)_(m)—NHC(O)OR¹³

—OCH₂CH₂CH₂CONHR¹³

—OCH₂CH₂NHCH₂(CHOH)₂CH₂CONHR¹³

—OCH₂CH₂CONH(CH₂[(CHOH)₂CH₂NH₂)]₂

—(CH₂)₄—NHCO₂R¹³

—(CH₂)₄—CONR¹³R¹³

—(CH₂)₄—CO₂R¹³

—OCH₂CH₂CONHSOCH₂CH₂N(CH₃)₂

—O—(CH₂)_(m)—C(═NH)—N(R¹³)₂

—(CH₂)_(n)—C(═NH)—N(R¹³)₂

—(CH₂)₃—NHCO₂R¹³—(CH₂)₃CONHCO₂R¹³

—O—(CH₂)_(m)—NH—NH—C(═NH)—N(R¹³)₂

—(CH₂)_(n)—NH—NH—C(═NH)-N(R¹³)₂ or

—O—CH₂—CHOH-CH₂—NH—C(═NH)—N(R¹³)₂.

The present invention also provides pharmaceutical compositions whichcontain a compound described above.

The present invention also provides a method of promoting hydration ofmucosal surfaces, comprising:

administering an effective amount of a compound represented by formula(I) to a mucosal surface of a subject.

The present invention also provides a method of restoring mucosaldefense, comprising:

topically administering an effective amount of compound represented byformula (I) to a mucosal surface of a subject in need thereof.

The present invention also provides a method of blocking ENaC,comprising:

contacting sodium channels with an effective amount of a compoundrepresented by formula (I).

The present invention also provides a method of promoting mucusclearance in mucosal surfaces, comprising:

administering an effective amount of a compound represented by formula(I) to a mucosal surface of a subject.

The present invention also provides a method of treating chronicbronchitis, comprising:

administering an effective amount of a compound represented by formula(I) to a subject in need thereof.

The present invention also provides a method of treating cysticfibrosis, comprising:

administering an effective amount of compound represented by formula (I)to a subject in need thereof.

The present invention also provides a method of treating rhinosinusitis,comprising:

administering an effective amount of a compound represented by a formula(I) to a subject in need thereof.

The present invention also provides a method of treating nasaldehydration, comprising:

administering an effective amount of a compound represented by formula(I) to the nasal passages of a subject in need thereof.

In a specific embodiment, the nasal dehydration is brought on byadministering dry oxygen to the subject.

The present invention also provides a method of treating sinusitis,comprising:

administering an effective amount of a compound represented by formula(I) to a subject in need thereof.

The present invention also provides a method of treating pneumonia,comprising:

administering an effective amount of a compound represented by formula(I) to a subject in need thereof.

The present invention also provides a method of preventingventilator-induced pneumonia, comprising:

administering an effective compound represented by formula (I) to asubject by means of a ventilator.

The present invention also provides a method of treating asthma,comprising:

administering an effective amount of a compound represented by formula(I) to a subject in need thereof.

The present invention also provides a method of treating primary ciliarydyskinesia, comprising:

administering an effective amount of a compound represented by formula(I) to a subject in need thereof.

The present invention also provides a method of treating otitis media,comprising:

administering an effective amount of a compound represented by formula(I) to a subject in need thereof.

The present invention also provides a method of inducing sputum fordiagnostic purposes, comprising:

administering an effective amount of compound represented by formula (I)to a subject in need thereof.

The present invention also provides a method of treating chronicobstructive pulmonary disease, comprising:

administering an effective amount of a compound represented by formula(I) to a subject in need thereof.

The present invention also provides a method of treating emphysema,comprising:

administering an effective amount of a compound represented by formula(I) to a subject in need thereof.

The present invention also provides a method of treating dry eye,comprising:

administering an effective amount of a compound represented by formula(I) to the eye of the subject in need thereof.

The present invention also provides a method of promoting ocularhydration, comprising:

administering an effective amount of a compound represented by formula(I) to the eye of the subject.

The present invention also provides a method of promoting cornealhydration, comprising:

administering an effective amount of a compound represented by formula(I) to the eye of the subject.

The present invention also provides a method of treating Sjögren'sdisease, comprising:

administering an effective amount of compound represented by formula (I)to a subject in need thereof.

The present invention also provides a method of treating vaginaldryness, comprising:

administering an effective amount of a compound represented by formula(I) to the vaginal tract of a subject in need thereof.

The present invention also provides a method of treating dry skin,comprising:

administering an effective amount of a compound represented by formula(I) to the skin of a subject in need thereof.

The present invention also provides a method of treating dry mouth(xerostomia), comprising:

administering an effective amount of compound represented by formula (I)to the mouth of the subject in need thereof.

The present invention also provides a method of treating distalintestinal obstruction syndrome, comprising:

administering an effective amount of compound represented by formula (I)to a subject in need thereof.

The present invention also provides a method of treating esophagitis,comprising:

administering an effective amount of a compound represented by formula(d) to a subject in need thereof.

The present invention also provides a method of treating constipation,comprising:

administering an effective amount of a compound represented by formula(I) to a subject in need thereof. In one embodiment of this method, thecompound is administered either orally or via a suppository or enema.

The present invention also provides a method of treating chronicdiverticulitis comprising:

administering an effective amount of a compound represented by formula(I) to a subject in need thereof.

The present invention also provides a method of treating hypertension,comprising administering the compound represented by formula (I) to asubject in need thereof.

The present invention also provides a method of reducing blood pressure,comprising administering the compound represented by formula (I) to asubject in need thereof.

The present invention also provides a method of treating edema,comprising administering the compound represented by formula (I) to asubject in need thereof.

The present invention also provides a method of promoting diuresis,comprising administering the compound represented by formula (I) to asubject in need thereof.

The present invention also provides a method of promoting natriuresis,comprising administering the compound represented by formula (I) to asubject in need thereof.

The present invention also provides a method of promoting saluresis,comprising administering the compound represented by formula (I) to asubject in need thereof.

DETAILED DESCRIPTION OF THE INVENTION

The present invention is based on the discovery that the compounds offormula (I) are more potent and/or, absorbed less rapidly from mucosalsurfaces, especially airway surfaces, and/or less reversible frominteractions with ENaC as compared to compounds such as amiloride,benzamil, and phenamil. Therefore, the compounds of formula (I) have alonger half-life on mucosal surfaces as compared to these compounds.

The present invention is also based on the discovery that certaincompounds embraced by formula (I) are converted in vivo into metabolicderivatives thereof that have reduced efficacy in blocking sodiumchannels as compared to the parent administered compound, after they areabsorbed from mucosal surfaces after administration. This importantproperty means that the compounds will have a lower tendency to causeundesired side-effects by blocking sodium channels located at untargetedlocations in the body of the recipient, e.g., in the kidneys.

The present invention is also based on the discovery that certaincompounds embraced by formula (I) are more soluble in aqueous solutions,especially in 0.12-0.9% saline, so that they can be convenientlyadministered to mucosal surfaces of a patient by suitable means such asa nebulizer, spay, mist or droplets. Therefore, the compounds of formula(I) are more soluble in aqueous solutions as compared to known compoundslacking an additional proatonateable nitrogen. The solubility of thecompounds in the saline solutions described above may be 0.1 to 7% byweight. The preferred range is 0.1 to 0.9% by weight.

In the compounds represented by formula (I), X may be hydrogen, halogen,trifluoromethyl, lower alkyl, lower cycloalkyl, unsubstituted orsubstituted phenyl, lower alkyl-thio, phenyl-lower alkyl-thio, loweralkyl-sulfonyl, or phenyl-lower alkyl-sulfonyl. Halogen is preferred.

Examples of halogen include fluorine, chlorine, bromine, and iodine.Chlorine and bromine are the preferred halogens. Chlorine isparticularly preferred. This description is applicable to the term“halogen” as used throughout the present disclosure.

As used herein, the term “lower alkyl” means an alkyl group having lessthan 8 carbon atoms. This range includes all specific values of carbonatoms and subranges there between, such as 1, 2, 3, 4, 5, 6, and 7carbon atoms. The term “alkyl” embraces all types of such groups, e.g.,linear, branched, and cyclic alkyl groups. This description isapplicable to the term “lower alkyl” as used throughout the presentdisclosure. Examples of suitable lower alkyl groups include methyl,ethyl, propyl, cyclopropyl, butyl, isobutyl, etc.

Substituents for the phenyl group include halogens. Particularlypreferred halogen substituents are chlorine and bromine.

Y may be hydrogen, hydroxyl, mercapto, lower alkoxy, lower alkyl-thio,halogen, lower alkyl, lower cycloalkyl, mononuclear aryl, or —N(R²)₂.The alkyl moiety of the lower alkoxy groups is the same as describedabove. Examples of mononuclear aryl include phenyl groups. The phenylgroup may be unsubstituted or substituted as described above. Thepreferred identity of Y is —N(R²)₂. Particularly preferred are suchcompounds where each R² is hydrogen.

R¹ may be hydrogen or lower alkyl. Hydrogen is preferred for R¹.

Each R² may be, independently, —R⁷, —(CH₂)_(m)—OR⁸, —(CH₂)_(m)—NR⁷R¹⁰,—(CH₂)_(n)(CHOR⁸)(CHOR⁸)_(n)—CH₂OR⁸, —(CH₂CH₂O)_(m)—R⁸,—(CH₂CH₂O)_(m)—CH₂CH₂NR⁷R¹⁰, —(CH₂)_(n)—C(═O)NR⁷R¹⁰,—(CH₂)_(n)-Z_(g)-R⁷,—(CH₂)_(m)—NR¹⁰—CH₂(CHOR⁸)(CHOR⁸)_(n)—CH₂OR⁸,—(CH₂)_(n)—CO₂R⁷, or

Hydrogen and lower alkyl, particularly C₁-C₃ alkyl are preferred for R².Hydrogen is particularly preferred.

R³ and R⁴ may be, independently, hydrogen, a group represented byformula (A), lower alkyl, hydroxy lower alkyl, phenyl, phenyl-loweralkyl, (halophenyl)-lower alkyl, lower- (alkylphenylalkyl), lower(alkoxyphenyl)-lower alkyl, naphthyl-lower alkyl, or pyridyl-loweralkyl, provided that at least one of R³ and R⁴ is a group represented byformula (A).

Preferred compounds are those where one of R³ and R⁴ is hydrogen and theother is represented by formula (A).

In formula (A), the moiety —(C(R^(L))₂)_(o)-x-(C(R^(L))₂)_(p)— definesan alkylene group bonded to the aromatic ring. The variables o and p mayeach be an integer from 0 to 10, subject to the proviso that the sum ofo and p in the chain is from 1 to 10. Thus, o and p may each be 0, 1, 2,3, 4, 5, 6, 7, 8, 9, or 10. Preferably, the sum of o and p is from 2 to6. In a particularly preferred embodiment, the sum of o and p is 4.

The linking group in the alkylene chain, x, may be, independently, O,NR¹⁰, C(═O), CHOH, C(═N—R¹⁰), CHNR⁷R¹⁰, or represents a single bond;

Therefore, when x represents a single bond, the alkylene chain bonded tothe ring is represented by the formula —(C(R^(L))₂)_(o+p)—, in which thesum o+p is from 1 to 10.

Each R^(L) may be, independently, —R⁷, —(CH₂)_(n)—OR⁸, —O—(CH₂)_(m)—OR⁸,—(CH₂)_(n)—NR⁷R¹⁰, —O—(CH₂)_(m)—NR⁷R¹⁰,—(CH₂)_(n)(CHOR⁸)(CHOR⁸)_(n)—CH₂OR⁸,—O—(CH₂)_(m)(CHOR⁸)(CHOR⁸)_(n)—CH₂OR⁸, —(CH₂CH₂O)_(m)—R⁸,—O—(CH₂CH₂O)_(m)—R⁸, —(CH₂CH₂O)_(m)—CH₂CH₂NR⁷R¹⁰,—O—(CH₂CH₂O)_(m)—CH₂CH₂NR⁷R¹⁰, —(CH₂)_(n)—C(═O)NR⁷R¹⁰,—O—(CH₂)_(m)—C(═O)NR⁷R¹⁰, —(CH₂)_(n)-(Z)_(g)-R⁷,—O—(CH₂)_(m)-(Z)_(g)-R⁷, —(CH₂)_(n)—NR¹⁰—CH₂(CHOR⁸)(CHOR⁸)_(n)—CH₂OR⁸,—O—(CH₂)_(m)—NR¹⁰—CH₂(CHOR⁸)(CHOR⁸)_(n)—CH₂OR⁸, —(CH₂)_(n)—CO₂R⁷,—O—(CH₂)_(m)—CO₂R⁷, —OSO₃H, —O-glucuronide, —O-glucose,

The preferred R^(L) groups include —H, —OH, —N(R⁷)₂, especially whereeach R⁷ is hydrogen.

In the alkylene chain in formula (A), it is preferred that when oneR^(L) group bonded to a carbon atoms is other than hydrogen, then theother R^(L) bonded to that carbon atom is hydrogen, i.e., the formula—CHR^(L)—. It is also preferred that at most two R^(L) groups in analkylene chain are other than hydrogen, where in the other R^(L) groupsin the chain are hydrogens. Even more preferably, only one R^(L) groupin an alkylene chain is other than hydrogen, where in the other R^(L)groups in the chain are hydrogens. In these embodiments, it ispreferable that x represents a single bond.

In another particular embodiment of the invention, all of the R^(L)groups in the alkylene chain are hydrogen. In these embodiments, thealkylene chain is represented by the formula

—(CH₂)_(o)-x-(CH₂)_(p)—.

There is one R⁵ present on the ring in formula (A). Each R⁵ may be,independently,

—(CH₂)_(n)—CO₂R¹³, Het-(CH₂)_(m)—CO₂R¹³, —(CH₂)_(n)-Z_(g)-CO₂R¹³,Het-(CH₂)_(m)-Z_(g)-CO₂R¹³, —(CH₂)_(n)—NR¹⁰—(CH₂)_(m)(CHOR⁸)_(n)—CO₂R³,Het-(CH₂)_(m)—NR¹⁰—(CH₂)_(m)(CHOR⁸)_(n)—CO₂R¹³,—(CH₂)_(n)(CHOR⁸)_(m)-CO₂R¹³, Het-(CH₂)_(m)—(CHOR⁸)_(m)—CO₂R¹³,—(CH₂)_(n)—(CHOR⁸)_(m)Z_(g)-CO₂R¹³, Het-(CH₂)_(n—(CHOR)⁸)_(m)-Z_(g)-CO₂R¹³, —(CH₂)_(n)-Z_(g)-(CH₂)_(m)—CO₂R¹³,—(CH₂)_(n)-Z_(g)-(CH₂)_(m)—CO₂R¹³,—(CH₂)_(n)-Z_(g)(CHOR⁸)_(m)-Z_(g)-CO₂R¹³,Het-(CH₂)_(n)-Z_(g)-(CHOR⁸)_(m)-Z_(g)-CO₂R¹³,—(CH₂)_(n)-CONH—C(═NR¹³)—NR¹³R¹³, Het-(CH₂)_(n)—CO—NH—C(═NR¹³)—NR³R¹³,—(CH₂)_(n)-Z_(g)-CONH—C(═NR¹³)—NR¹³R¹³,Het-(CH₂)_(n)-Z_(g)-CONH—C(═NR¹³)—NR¹³R¹³,—(CH₂)_(n)-NR¹⁰—(CH₂)_(m)(CHOR⁸)_(n)—CONH—C(═NR¹³)—NR¹³R¹³,Het-(CH₂)_(n)—NR¹⁰—(CH₂)_(m)(CHOR⁸)_(n)—CONH—C(═NR¹³)—NR¹³R¹³,—(CH₂)_(n)—(CHOR⁸)_(m)—CONH—C(═NR¹³)—NR¹³R¹³,Het-(CH₂)_(n)—(CHOR⁸)_(m)—CONH—C(═NR¹³)—NR¹³R¹³,—(CH₂)_(n)—(CHOR⁸)_(m)-Z_(g)-CONH—C(═NR¹³)—NR¹³R¹³,Het-(CH₂)_(n)—(CHOR⁸)_(m)-Z_(g)-CONH—C(═NR¹³)—NR¹³R¹³,—(CH₂)_(n)-Z_(g)-(CH₂)_(m)CONH—C(═NR¹³)—NR¹³R¹³,Het-(CH₂)_(n)-Z_(g)-(CH₂)_(m)CONH—C(═NR¹³)—NR¹³R¹³,—(CH₂)_(n)-Z_(g)-(CHOR⁸)_(m)-Z_(g)-CONH—C(═NR¹³)—NR¹³R¹³,Het-(CH₂)_(n)-Z_(g)-(CHOR⁸)_(m)-Z_(g)-CONH—C(═NR¹³)—NR¹³R¹³,—(CH₂)_(n)—CONR⁷—CONR¹³R¹³, Het-(CH₂)_(n)—CONR⁷—CONR¹³R¹³,—(CH₂)_(n)-Z_(g)-CONR⁷-CONR¹³R¹³, —(CH₂)_(n)-Z_(g)-CONR⁷—CONR¹³R¹³,—(CH₂)_(n)—NR¹⁰—(CH₂)_(m)(CHOR⁸)_(n)—CONR⁷—CONR¹³R¹³, Het-(CH₂)_(n)—NR¹⁰—(CH₂)_(m)(CHOR⁸)_(n)—CONR⁷—CONR¹³R¹³,—(CH₂)_(n)—(CHOR⁸)_(m)—CONR⁷—CONR¹³R¹³,Het-(CH₂)_(n)—(CHOR⁸)_(m)—CONR⁷—CONR¹³R¹³,—(CH₂)_(n)—(CHOR⁸)_(m)-Z_(g)-CONR⁷—CONR¹³R¹³,Het-(CH₂)_(n)—(CHOR⁸)_(m)-Z_(g)-CNR⁷—CONR¹³R¹³,—(CH₂)_(n)-Z_(g)-(CH₂)_(m)CONR⁷—CONR¹³R¹³,Het-(CH₂)_(n)-Z_(g)-(CH₂)_(m)CONR⁷—CONR¹³R¹³,—(CH₂)_(n)-Z_(g)(CHOR⁸)_(m)-Z_(g)-CONR⁷—CONR¹³R¹³,Het-(CH₂)_(n)-Z_(g)(CHOR⁸)_(m)-Z_(g)-CONR⁷—CONR¹³R¹³,—(CH₂)_(n)—CONR⁷SO₂NR¹³R¹³, Het-(CH₂)_(m)—CONR⁷SO₂NR¹³R¹³,—(CH₂)_(n)-Z_(g)-CONR⁷SO₂NR¹³R¹³, Het-(CH₂)_(m)-Z_(g)-CONR⁷SO₂NR¹³R¹³,—(CH₂)_(n)—NR¹⁰—(CH₂)_(m)(CHOR⁸)_(n)—CONR⁷SO₂NR¹³R¹³,Het-(CH₂)_(m)—NR¹⁰—(CH₂)_(m)(CHOR⁸)_(n)—CONR⁷SO₂NR¹³R¹³,—(CH₂)_(n)—(CHOR⁸)_(m)—CONR⁷SO₂NR¹³R¹³,Het-(CH₂)_(m)—(CHOR⁸)_(m)—CONR⁷SO₂NR¹³R¹³,—(CH₂)_(n)—(CHOR⁸)_(m)-Z_(g)-CONR⁷SO₂NR¹³R¹³,Het-(CH₂)_(n)-(CHOR⁸)_(m)-Z_(g)-CONR⁷SO₂NR¹³R¹³,—(CH₂)_(n)-Z_(g)-(CH₂)_(m)CONR⁷SO₂NR¹³R¹³,Het-(CH₂)_(n)-Z_(g)-(CH₂)_(m)CONR⁷SO₂NR¹³R¹³—(CH₂)_(n)-Z_(g)-(CHOR⁸)_(m)-Z_(g)-CONR⁷SO₂NR¹³R¹³,Het-(CH₂)_(n)-Z_(g)-(CHOR⁸)_(m)-Z_(g)-CONR⁷SO₂NR³R¹³,—(CH₂)_(n)—SO₂NR¹³RN¹³, Het-(CH₂)_(m)—SO₂NR¹³R¹³,—(CH₂)_(n)-Z_(g)-SO₂NR¹³R¹³, Het-(CH₂)_(m)-Z_(g)-SO₂NR¹³R¹³,(CH₂)_(n)NR¹⁰—(CH₂)_(m)(CHOR⁸)_(n)—SO₂NR₁₃R¹³,Het-(CH₂)_(m)—NR¹⁰—(CH₂)_(m)(CHOR⁸)_(n)—SO₂NR¹³R¹³,—(CH₂)_(n)—(CHOR⁸)_(m)—SO₂NR¹³R¹³, Het-(CH₂)_(m)—(CHOR⁸)_(m)—SO₂NR¹³R¹³,—(CH₂)_(n)—(CHOR⁸)_(m)-Z_(g)-SO₂NR¹³R¹³,Het-(CH₂)_(n)—(CHOR⁸)_(m)-Z_(g)-SO₂NR¹³R¹³,—(CH₂)_(n)-Z_(g)-(CH₂)_(m)SO₂NR¹³R¹³,Het-(CH₂)_(n)-Z_(g)-(CH₂)_(m)SO₂NR¹³R¹³,—(CH₂)_(n)-Z_(g)-(CHOR⁸)_(m)-Z_(g)-SO₂NR¹³R¹³,Het-(CH₂)_(n)-Z_(g)-(CHOR⁸)_(m)-Z_(g)-SO₂NR¹³R¹³, —(CH₂)_(n)—CONR¹³R¹³,Het-(CH₂)_(m)—CONR¹³R¹³, (CH₂)_(n)Z_(g)CONR¹³R¹³,Het-(CH₂)_(m)-Z_(g)-CONR¹³R¹³,—(CH₂)_(n)—NR¹⁰—(CH₂)_(m)(CHOR⁸)_(n)-CONR¹³R¹³,Het-(CH₂)_(m)—NR¹⁰—(CH₂)_(m)(CHOR⁸)_(n)—CONR¹³R¹³,—(CH₂)_(n)—(CHOR⁸)_(m)—CONR¹³R¹³, Het-(CH₂)_(m)—(CHOR⁸)_(m)—CONR¹³R¹³,—(CH₂)_(n)—(CHOR⁸)_(m)-Z_(g)-CONR¹³R¹³,Het-(CH₂)_(n)—(CHOR⁸)_(m)-Z_(g)-CONR¹³R¹³,—(CH₂)_(n)-Z_(g)-(CH₂)_(m)CONR¹³R¹³,Het-(CH₂)_(n)-Z_(g)-(CH₂)_(m)CONR¹³R¹³,—(CH₂)_(n)-Z_(g)-(CHOR⁸)_(m)-Z_(g)-CONR¹³R¹³,Het-(CH₂)_(n)-Z_(g)-(CHOR⁸)_(m)-Z_(g)-CONR¹³R¹³, —(CH₂)_(n)—CONR⁷COR¹³,Het-(CH₂)_(m)—CONR⁷COR¹³, —(CH₂)_(n)-Z_(g)-CONR⁷COR¹³,Het-(CH₂)_(m)-Z_(g)-CONR⁷COR¹³,—(CH₂)_(n)—NR¹⁰—(CH₂)_(m)(CHOR⁸)_(n)—CONR⁸)_(n)—CONR⁷COR¹³,Het-(CH₂)_(m)NR¹⁰—(CH₂)_(m)(CHOR⁸)_(n)—CONR⁷COR¹³,—(CH₂)_(n)—(CHOR⁸)_(m)—CONR⁷COR¹³, Het-(CH₂)_(m)(CHOR⁸)_(m)—CONR⁷COR¹³,—(CH₂)_(n)—(CHOR⁸)_(m)-Z_(g)-CONR⁷COR¹³,Het-(CH₂)_(n)—(CHOR⁸)_(m)-Z_(g)-CONR⁷COR¹³,—(CH₂)_(n)-Z_(g)-(CH₂)_(m)CONR⁷COR¹³,—(CH₂)_(n)-Z_(g)-(CH₂)_(m)CONR⁷COR¹³,Het-(CH₂)_(n)-Z_(g)-(CHOR⁸)_(m)-Z_(g)-CONR⁷COR¹³,—(CH₂)_(n)—CONR⁷CO₂R¹³, —(CH₂)_(n)-Z_(g)-CONR⁷CO₂R¹³,Het-(CH₂)_(m)-Z_(g)-CONR⁷CO₂R¹³,—(CH₂)_(n)—NR¹⁰—(CH₂)_(m)(CHOR⁸)_(n)-CONR⁷CO₂R¹³,Het-(CH₂)_(m)—NR¹⁰—(CH₂)_(m)(CHOR⁸)_(n)—CONR⁷CO₂R¹³,—(CH₂)_(n)(CHOR⁸)_(m)—CONR⁷CO₂R¹³,Het-(CH₂)_(m)—(CHOR⁸)_(m)—CONR⁷CO₂R¹³,(CH₂)_(n)—(CHOR⁸)_(m)-Z_(g)-CONR⁷CO₂R¹³,Het-(CH₂)_(n)—(CHOR⁸)_(m)-Z_(g)-CONR⁷CO₂R¹³,—(CH₂)_(n)-Z_(g)-(CH₂)_(m)CONR⁷CO₂R¹³,Het-(CH₂)_(n)-Z_(g)-(CH₂)_(m)CONR⁷CO₂R¹³,—(CH₂)_(n)-Z_(g)-(CHOR⁸)_(m)-Z_(g)-CONR⁷CO₂R¹³,Het-(CH₂)_(n)-Z_(g)-(CHOR⁸)_(m)-Z_(g)-CONR⁷CO₂R¹³,—(CH₂)_(n)—NH—C(═NR¹³)—NR¹³R¹³, Het-(CH₂)_(m)—NH—C(═NR¹³)—NR¹³R¹³,—(CH₂)_(n)-Z_(g)-NH—C(═NR¹³)—NR¹³R¹³,Het-(CH₂)_(m)-Z_(g)-NH—C(═NR¹³)—NR¹³R¹³,—(CH₂)_(n)—NR¹⁰—(CH₂)_(m)(CHOR⁸)_(n)—NH—C(═NR¹³)—NR¹³R¹³,Het-(CH₂)_(m)—NR¹⁰—(CH₂)_(m)(CHOR⁸)_(n)—NH—C(═NR¹³)—NR¹³R¹³,—(CH₂)_(n)—(CHOR⁸)_(m)-NH—C(═NR¹³)—NR¹³R¹³,Het-(CH₂)_(m)—(CHOR⁸)_(m)—NH—C(═NR¹³)—NR¹³R¹³,—(CH₂)_(n)—(CHOR⁸)_(m)-Z_(g)-NH—C(═NR¹³)—NR¹³R¹³,Het-(CH₂)_(n)-(CHOR⁸)_(m)-Z_(g)-NH—C(═NR¹³)—NR¹³R¹³,—(CH₂)_(n)-Z_(g)-(CH₂)_(m)NH—C(═NR¹³)—NR¹³R¹³,Het-(CH₂)_(n)-Z_(g)-(CH₂)_(m)NH—C(═NR¹³)—NR¹³R¹³,—(CH₂)_(n)-Z_(g)-(CHOR⁸)_(m)-Z_(g)-NH—C(═NR¹³)—NR¹³R¹³,Het-(CH₂)_(n)-Z_(g)-(CHOR⁸)_(m)-Z_(g)-NH—C(═NR¹³)—NR¹³R¹³,—(CH₂)_(n)—C(═NR¹³)—NR¹³R¹³, Het-(CH₂)_(m)—C(═NH)—NR¹³R¹³,—(CH₂)_(n)-Z_(g)-C(═NR¹³)—NR¹³R¹³, -Het-(CH₂)_(m)-Z_(g)-C(═NH)—NR¹³R¹³,—(CH₂)_(n)—NR¹⁰—(CH₂)_(m)(CHOR⁸)_(n)—C(═NR¹³)—NR¹³R¹³,Het-(CH₂)_(m)—NR¹⁰—C(CH₂)_(m)(CHOR⁸)_(n)—C(═NR¹³)—NR¹³R¹³,—(CH₂)_(n)—(CHOR⁸)_(m)—C(═NR¹³)—NR¹³R¹³,Het-(CH₂)_(m)—(CHOR⁸)_(m)—C(═NR¹³)—NR¹³R¹³,—(CH₂)_(n)—(CHOR⁸)_(m)-Z_(g)-C(═NR¹³)—NR¹³R¹³,Het-(CH₂)_(n)—(CHOR⁸)_(m)-Z_(g)-C(═NR¹³)—NR¹³R¹³,—(CH₂)_(n)-Z_(g)-(CH₂)_(m)—C(═NHC(═NR¹³)—NR¹³R¹³,Het-(CH₂)_(n)-Z_(g)-(CH₂)_(m)—C(═NR¹³)—NR¹³R¹³,—(CH₂)_(n)-Z_(g)-(CHOR⁸)_(m)-Z_(g)-C(═NR¹³)—NR¹³R¹³,Het-(CH₂)_(n)-Z_(g)-(CHOR⁸)_(m)-Z_(g)-C(═NR¹³)—NR¹³R¹³;

with the proviso wherein when two —CH₂OR⁸ groups are located 1,2- or1,3- with respect to each other the R⁸ groups may be joined to form acyclic mono- or di-substituted 1,3-dioxane or 1,3-dioxolane;

In a preferred embodiment, each —(CH₂)_(n)-Z_(g)-C(═NH)—NR¹³R¹³ fallswithin the scope of the structures described above and is,independently, —(CH₂)_(n)—CHNH(C═N)—NR¹³R¹³.

In another preferred embodiment, each Het-(CH₂)_(m)—NH—C(═NH)—NR¹³R¹³falls within the scope of the structures described above and is,independently, —(CH₂)_(n)—NH—C(═NH)NHR¹³.

In another preferred embodiment, each—(CH₂)_(n)-Z_(g)-(CHOR⁸)_(m)-Z_(g)-CONR¹³R¹³ falls within the scope ofthe structures described above and is, independently,—(CH₂)_(n)—CONHCH₂(CHOH)_(m)—CONHR¹³.

In another preferred embodiment, eachHet-(CH₂)_(n)-Z_(g)-(CHOR⁸)_(m)-Z_(g)-CONR¹³R¹³ falls within the scopeof the structures described above and is, independently,—NH—C(═O)—CH₂—(CHOH)_(n)CH₂CONR¹³R¹³.

In another a preferred embodiment, eachHet-(CH₂)_(m)-Z_(g)-C(═NH)—NR¹³R¹³ falls within the scope of thestructures described above and is, independently,—O—(CH₂)_(m)—NH—C(═NH)—N(R¹³)₂.

In another a preferred embodiment, each Het-(CH₂)_(m)-Z_(g)-CONR¹³R¹³falls within the scope of the structures described above and is,independently, —O—(CH₂)_(m)—CHNH₂—CO₂NR¹³R¹³.

In another preferred embodiment, each R⁵ falls within the scope of thestructures described above and is, independently,

—O—CH₂CHOHCH₂CONR¹³R¹³

—OCH2CHOHCH₂CO₂R¹³ OCH₂CH₂CONR¹³R¹³

—OCH₂CH₂NHCOR¹³

—CH₂CH₂CONR¹³R¹³

—OCH₂CH₂CONR¹³R¹³ O—(CH₂)_(m)—CO₂R¹³

—(CH₂)_(m)—CO₂R¹³

—OCH₂CH₂CO₂R¹³

—OCH₂CO₂R¹³

—O—(CH₂)_(m)—NH—C(═NH)—NR¹³)₂

—(CH₂)_(n)—NH—C(═NH)—N(R¹³)₂

—NHCH₂(CHOH)₂—CONR¹³R¹³

—OCH₂CO₂R¹³

—NHSO₂(CH₂)₂CONR¹³R¹³

—(CH₂)_(m)—NH—C(═O)—OR¹³

—O—(CH₂)_(m)—NH—C(═O)—OR¹³

—(CH₂)_(n)—NH—C(═O)—R¹³

—O—(CH₂)_(m)—NH—C(═O)—R¹³

—O—CH₂C(═ONR¹³R¹³

—CH₂NCO₂R¹³

—NHCO₂R¹³

—OCH₂CH₂CH₂CH₂CONR¹³R¹³

—SO₂CH₂CH₂CONR¹³R¹³

—OCH₂CH₂CHOHCH₂CONR¹³R¹³

—OCH₂CH₂NHCO₂R¹³

—NH—C(═NH2)—NR¹³R¹³

—OCH₂(α-CHOH)₂—CONR¹³R¹³

—OCH₂CHOHCH₂CONHR¹³

—(CH₂)_(m)—CHOH—CH₂—NHCO₂R¹³

—O—(CH₂)_(m)—CHOH—CH₂—CO₂R¹³

—(CH₂)_(m)—NHC(O)OR¹³

—O—(CH₂)_(m)—NHC(O)OR¹³

—OCH₂CH₂CH₂CONHR¹³

—OCH₂CH₂NHCH₂(CHOH)₂CH₂CONHR¹³

—OCH₂CH₂CONH(CH₂[(CHOH)₂CH₂NH₂)]₂

—(CH₂)₄—NHCO₂R¹³

—(CH₂)₄—CONR¹³R¹³

—(CH₂)₄—CO₂ ^(R) ¹³

—OCH₂CH₂CONHSOCH₂CH₂N(CH₃)₂

—O—(CH₂)_(m)—C(═NH)—N(R¹³)₂

—(CH₂)_(n)—C(═NH)—N(R¹³)₂

—(CH₂)₃—NHCO₂R¹³—(CH₂)₃CONHCO₂R¹³

—O—(CH₂)_(m)—NH—NH—C(═NH)—N(R¹³)₂

—(CH₂)_(n)—NH—NH—C(═NH)—N(R¹³)₂, or

—O—CH₂—CHOH—CH₂—NH—C(═NH)—N(R¹³)₂;

There are four R⁶ groups present on the ring in formula (A). Each R⁶ maybe each, independently, —R⁷, —OR¹¹, —N(R⁷)₂, —(CH₂)_(m)—OR⁸,

—O—(CH₂)_(m)—OR⁸, —(CH₂)_(n)—NR⁷R¹⁰, —O—(CH₂)_(m)—NR⁷R¹⁰,

—(CH₂)_(m)(CHOR⁸)(CHOR⁸)_(n)—CH₂OR⁸,—O—(CH₂)_(m)(CHOR⁸)(CHOR⁸)_(n)—CH₂OR⁸,

—(CH₂CH₂O)_(m)—R⁸, —O—(CH₂CH₂O)_(m)—R⁸, —(CH₂CH₂O)_(m)—CH₂CH₂NR⁷R¹⁰,

—O—(CH₂CH₂O)_(m)—CH₂CH₂NR⁷R¹⁰, —(CH₂)_(n)—C(═O)NR⁷R¹⁰,

—O—(CH₂)_(m)—C(═O)NR⁷R¹⁰, —(CH₂)_(n)-(Z)_(g)-R⁷,—O—(CH₂)_(m)-(Z)_(g)-R⁷,

—(CH₂)_(n)—NR¹⁰—CH₂(CHOR⁸)(CHOR⁸)_(n)—CH₂OR⁸,

—O—(CH₂)_(m)—NR¹⁰—CH₂(CHOR⁸)(CHOR⁸)_(n)—CH₂OR⁸,

—(CH₂)_(n)—CO₂R⁷, —O—(CH₂)_(m)—CO₂R⁷, —OSO₃H, —O-glucuronide,—O-glucose, or

In addition, one of more of the R⁶ groups can be one of the R⁵ groupswhich fall within the broad definition of R⁶ set forth above.

When two R⁶ are —OR¹¹ and are located adjacent to each other on a phenylring, the allyl moieties of the two R⁶ groups may be bonded together toform a methylenedioxy group, i.e., a group of the formula —O—CH₂—O—.

As discussed above, R⁶ may be hydrogen. Therefore, 1, 2, 3, or 4 R⁶groups may be other than hydrogen. Preferably at most 3 of the R⁶ groupsare other than hydrogen.

Each g is, independently, an integer from 1 to 6. Therefore, each g maybe 1, 2, 3, 4, 5, or 6.

Each m is an integer from 1 to 7. Therefore, each m may be 1, 2, 3, 4,5, 6, or 7.

Each n is an integer from 0 to 7. Therefore, each n maybe 0, 1, 2, 3, 4,5, 6, or 7.

Each Q in formula (A) is C—R⁵, C—R⁶, or a nitrogen atom, where at mostthree Q in a ring are nitrogen atoms. Thus, there may be 1, 2, or 3nitrogen atoms in a ring. Preferably, at most two Q are nitrogen atoms.More preferably, at most one Q is a nitrogen atom. In one particularembodiment, the nitrogen atom is at the 3-position of the ring. Inanother embodiment of the invention, each Q is either C—R⁵ or C—R⁶,i.e., there are no nitrogen atoms in the ring.

More specific examples; of suitable groups represented by formula (A)are shown in formulas (B)-(E) below:

where o, x, p, R⁵, and R⁶, are as defined above;

where n is an integer from 1 to 10 and R⁵ is as defined above;

where n is an integer from 1 from 10 and R⁵ is as defined above;

where o, x, p, and R⁵ are as defined above.

In a preferred embodiment of the invention, Y is —NH₂.

In another preferred embodiment, R² is hydrogen.

In another preferred embodiment, R¹ is hydrogen.

In another preferred embodiment, X is chlorine.

In another preferred embodiment, R³ is hydrogen.

In another preferred embodiment, R^(L) is hydrogen.

In another preferred embodiment, o is 4.

In another preferred embodiment, p is 0.

In another preferred embodiment, the sum of o and p is 4.

In another preferred embodiment, x represents a single bond.

In another preferred embodiment, R⁶ is hydrogen.

In another preferred embodiment, at most one Q is a nitrogen atom.

In another preferred embodiment, no Q is a nitrogen atom.

In a preferred embodiment of the present invention:

X is halogen;

Y is —N(R⁷)₂;

R¹ is hydrogen or C₁-C₃ alkyl;

R¹ is —R⁷, —OR⁷, CH₂O⁷, or —CO₂R⁷;

R³ is a group represented by formula (A); and

R⁴ is hydrogen, a group represented by formula (A), or lower alkyl;

In another preferred embodiment of the present invention:

X is chloro or bromo;

Y is —N(R⁷)₂;

R² is hydrogen or C₁-C₃ alkyl;

at most three R⁶ are other than hydrogen as described above;

at most three R^(L) are other than hydrogen as described above; and

at most 2 Q are nitrogen atoms.

In another preferred embodiment of the present invention:

Y is —NH₂;

In another preferred embodiment of the present invention:

R⁴ is hydrogen;

at most one R^(L) is other than hydrogen as described above;

at most two R⁶ are other than hydrogen as described above; and

at most 1 Q is a nitrogen atom.

In another preferred embodiment of the present invention the compound offormula (1) is represented by the formula:

In another preferred embodiment of the present invention the compound offormula (1) is represented by the formula:

In another preferred embodiment of the present invention the compound offormula (1) is represented by the formula:

In another preferred embodiment of the present invention the compound offormula (1) is represented by the formula:

In another preferred embodiment of the present invention the compound offormula (1) is represented by the formula:

In another preferred embodiment of the present invention the compound offormula (1) is represented by the formula:

In another preferred embodiment of the present invention the compound offormula (1) is represented by the formula:

In another preferred embodiment of the present invention the compound offormula (1) is represented by the formula:

In another preferred embodiment of the present invention the compound offormula (1) is represented by the formula:

In another preferred embodiment of the present invention the compound offormula (1) is represented by the formula:

In another preferred embodiment of the present invention the compound offormula (1) is represented by the formula:

In another preferred embodiment of the present invention the compound offormula (1) is represented by the formula:

In another preferred embodiment of the present invention the compound offormula (1) is represented by the formula:

In another preferred embodiment of the present invention the compound offormula (1) is represented by the formula:

In another preferred embodiment of the present invention the compound offormula (1) is represented by the formula:

In another preferred embodiment of the present invention the compound offormula (1) is represented by the formula:

In another preferred embodiment of the present invention the compound offormula (1) is represented by the formula:

In another preferred embodiment of the present invention the compound offormula (1) is represented by the formula:

In another preferred embodiment of the present invention the compound offormula (1) is represented by the formula:

In another preferred embodiment of the present invention the compound offormula (1) is represented by the formula:

In another preferred embodiment of the present invention the compound offormula (1) is represented by the formula:

In another preferred embodiment of the present invention the compound offormula (1) is represented by the formula:

In another preferred embodiment of the present invention the compound offormula (1) is represented by the formula:

In another preferred embodiment of the present invention the compound offormula (1) is represented by the formula:

In another preferred embodiment of the present invention the compound offormula (1) is represented by the formula:

In another preferred embodiment of the present invention the compound offormula (1) is represented by the formula:

In another preferred embodiment of the present invention the compound offormula (1) is represented by the formula:

In another preferred embodiment of the present invention the compound offormula (1) is represented by the formula:

In another preferred embodiment of the present invention the compound offormula (1) is represented by the formula:

In another preferred embodiment of the present invention the compound offormula (1) is represented by the formula:

In another preferred embodiment of the present invention the compound offormula (1) is represented by the formula:

In another preferred embodiment of the present invention the compound offormula (1) is represented by the formula:

In another preferred embodiment of the present invention the compound offormula (1) is represented by the formula:

In another preferred embodiment of the present invention the compound offormula (1) is represented by the formula:

In another preferred embodiment of the present invention the compound offormula (1) is represented by the formula:

In another preferred embodiment of the present invention the compound offormula (1) is represented by the formula:

In another preferred embodiment of the present invention the compound offormula (1) is represented by the formula:

In another preferred embodiment of the present invention the compound offormula (1) is represented by the formula:

In another preferred embodiment of the present invention the compound offormula (1) is represented by the formula:

In another preferred embodiment of the present invention the compound offormula (1) is represented by the formula:

In another preferred embodiment of the present invention the compound offormula (1) is represented by the formula:

In another preferred embodiment of the present invention the compound offormula (1) is represented by the formula:

In another preferred embodiment of the present invention the compound offormula (1) is represented by the formula:

In another preferred embodiment of the present invention the compound offormula (1) is represented by the formula:

In another preferred embodiment of the present invention the compound offormula (1) is represented by the formula:

In another preferred embodiment of the present invention the compound offormula (1) is represented by the formula:

In another preferred embodiment of the present invention the compound offormula (1) is represented by the formula:

In another preferred embodiment of the present invention the compound offormula (1) is represented by the formula:

In another preferred embodiment of the present invention the compound offormula (1) is represented by the formula:

In another preferred embodiment of the present invention the compound offormula (1) is represented by the formula:

In another preferred embodiment of the present invention the compound offormula (1) is represented by the formula:

In another preferred embodiment of the present invention the compound offormula (1) is represented by the formula:

In another preferred embodiment of the present invention the compound offormula (1) is represented by the formula:

In another preferred embodiment of the present invention the compound offormula (1) is represented by the formula:

In another preferred embodiment of the present invention the compound offormula (1) is represented by the formula:

In another preferred embodiment of the present invention the compound offormula (1) is represented by the formula:

In another preferred embodiment of the present invention the compound offormula (1) is represented by the formula:

In another preferred embodiment of the present invention the compound offormula (1) is represented by the formula:

In another preferred embodiment of the present invention the compound offormula (1) is represented by the formula:

In another preferred embodiment of the present invention the compound offormula (1) is represented by the formula:

In another preferred embodiment of the present invention the compound offormula (1) is represented by the formula:

In another preferred embodiment of the present invention the compound offormula (1) is represented by the formula:

In another preferred embodiment of the present invention the compound offormula (1) is represented by the formula:

In another preferred embodiment of the present invention the compound offormula (1) is represented by the formula:

In another preferred embodiment of the present invention the compound offormula (1) is represented by the formula:

In another preferred embodiment of the present invention the compound offormula (1) is represented by the formula:

In another preferred embodiment of the present invention the compound offormula (1) is represented by the formula:

In another preferred embodiment of the present invention the compound offormula (1) is represented by the formula:

In another preferred embodiment of the present invention the compound offormula (1) is represented by the formula:

In another preferred embodiment of the present invention the compound offormula (1) is represented by the formula:

In another preferred embodiment of the present invention the compound offormula (1) is represented by the formula:

In another preferred embodiment of the present invention the compound offormula (1) is represented by the formula:

In another preferred embodiment of the present invention the compound offormula (1) is represented by the formula:

In another preferred embodiment of the present invention the compound offormula (1) is represented by the formula:

The compounds of formula (I) may be prepared and used as the free base.Alternatively, the compounds may be prepared and used as apharmaceutically acceptable salt. Pharmaceutically acceptable salts aresalts that retain or enhance the desired biological activity of theparent compound and do not impart undesired toxicological effects.Examples of such salts are (a) acid addition salts formed with inorganicacids, for example, hydrochloric acid, hydrobromic acid, sulfuric acid,phosphoric acid, nitric acid and the like; (b) salts formed with organicacids such as, for example, acetic acid, oxalic acid, tartaric acid,succinic acid, maleic acid, fumaric acid, gluconic acid, citric acid,malic acid, ascorbic acid, benzoic acid, tannic acid, palmitic acid,alginic acid, polyglutamic acid, naphthalenesulfonic acid,methanesulfonic acid, p-toluenesulfonic acid, naphthalenedisulfonicacid, polygalacturonic acid, malonic acid, sulfosalicylic acid, glycolicacid, 2-hydroxy-3-naphthoate, pamoate, salicylic acid, stearic acid,phthalic acid, mandelic acid, lactic acid and the like; and (c) saltsformed from elemental anions for example, chlorine, bromine, and iodine.

It is to be noted that all enantiomers, diastereomers, and racemicmixtures of compounds within the scope of formula (I) are embraced bythe present invention. All mixtures of such enantiomers anddiastereomers are within the scope of the present invention.

Without being limited to any particular theory, it is believed that thecompounds of formula (I) function in vivo as sodium channel blockers. Byblocking epithelial sodium channels present in mucosal surfaces thecompounds of formula (I) reduce the absorption of water by the mucosalsurfaces. This effect increases the volume of protective liquids onmucosal surfaces, rebalances the system, and thus treats disease.

The present invention also provides methods of treatment that takeadvantage of the properties of the compounds of formula (I) discussedabove. Thus, subjects that may be treated by the methods of the presentinvention include, but are not limited to, patients afflicted withcystic fibrosis, primary ciliary dyskinesia, chronic bronchitis, chronicobstructive airway disease, artificially ventilated patients, patientswith acute pneumonia, etc. The present invention may be used to obtain asputum sample from a patient by administering the active compounds to atleast one lung of a patient, and then inducing or collecting a sputumsample from that patient. Typically, the invention will be administeredto respiratory mucosal surfaces via aerosol (liquid or dry powders) orlavage.

Subjects that may be treated by the method of the present invention alsoinclude patients being administered supplemental oxygen nasally (aregimen that tends to dry the airway surfaces); patients afflicted withan allergic disease or response (e.g., an allergic response to pollen,dust, animal hair or particles, insects or insect particles, etc.) thataffects nasal airway surfaces; patients afflicted with a bacterialinfection e.g., staphylococcus infections such as Staphylococcus aureusinfections, Hemophilus influenza infections, Streptococcus pneumoniaeinfections, Pseudomonas aeuriginosa infections, etc.) of the nasalairway surfaces; patients afflicted with an inflammatory disease thataffects nasal airway surfaces; or patients afflicted with sinusitis(wherein the active agent or agents are administered to promote drainageof congested mucous secretions in the sinuses by administering an amounteffective to promote drainage of congested fluid in the sinuses), orcombined, Rhinosinusitis. The invention may be administered torhino-sinal surfaces by topical delivery, including aerosols and drops.

The present invention may be used to hydrate mucosal surfaces other thanairway surfaces. Such other mucosal surfaces include gastrointestinalsurfaces, oral surfaces, genito-urethral surfaces, ocular surfaces orsurfaces of the eye, the inner ear and the middle ear. For example, theactive compounds of the present invention may be administered by anysuitable means, including locally/topically, orally, or rectally, in aneffective amount.

The compounds of the present invention are also useful for treating avariety of functions relating to the cardiovascular system. Thus, thecompounds of the present invention are useful for use asantihypertensive agents. The compounds may also be used to reduce bloodpressure and to treat edema. In addition, the compounds of the presentinvention are also useful for promoting diuresis, natriuresis, andsaluresis. The compounds may be used alone or in combination with betablockers, ACE inhibitors, HMGCoA, reductase inhibitors, calcium channelblockers and other cardiovascular agents to treat hypertension,congestive heart failure and reduce cardiovascular mortality.

The present invention is concerned primarily with the treatment of humansubjects, but may also be employed for the treatment of other mammaliansubjects, such as dogs and cats, for veterinary purposes.

As discussed above, the compounds used to prepare the compositions ofthe present invention may be in the form of a pharmaceuticallyacceptable free base. Because the free base of the compound is generallyless soluble in aqueous solutions than the salt, free base compositionsare employed to provide more sustained release of active agent to thelungs. An active agent present in the lungs in particulate form whichhas not dissolved into solution is not available to induce aphysiological response, but serves as a depot of bioavailable drug whichgradually dissolves into solution.

Another aspect of the present invention is a pharmaceutical composition,comprising a compound of formula (I) in a pharmaceutically acceptablecarrier (e.g., an aqueous carrier solution). In general, the compound offormula (I) is included in the composition in an amount effective toinhibit the reabsorption of water by mucosal surfaces.

The compounds of the present invention may also be used in conjunctionwith a P2Y2 receptor agonist or a pharmaceutically acceptable saltthereof (also sometimes referred to as an “active agent” herein). Thecomposition may further comprise a P2Y2 receptor agonist or apharmaceutically acceptable salt thereof (also sometimes referred to asan “active agent” herein). The P2Y2 receptor agonist is typicallyincluded in an amount effective to stimulate chloride and watersecretion by airway surfaces, particularly nasal airway surfaces.Suitable P2Y2 receptor agonists are described in columns 9-10 of U.S.Pat. No. 6,264,975, U.S. Pat. No. 5,656,256, and U.S. Pat. No.5,292,498, each of which is incorporated herein by reference.

Bronchodiloators can also be used in combination with compounds of thepresent invention. These bronchodilators include, but are not limitedto, β-adrenergic agonists including but not limited to epinephrine,isoproterenol, fenoterol, albutereol, terbutalin, pirbuterol,bitolterol, metaproterenol, iosetharine, salmeterol xinafoate, as wellas anticholinergic agents including but not limited to ipratropiumbromide, as well as compounds such as theophylline and aminophylline.These compounds may be administered in accordance with known techniques,either prior to or concurrently with the active compounds describedherein.

Another aspect of the present invention is a pharmaceutical formulation,comprising an active compound as described above in a pharmaceuticallyacceptable carrier (e.g., an aqueous carrier solution). In general, theactive compound is included in the composition in an amount effective totreat mucosal surfaces, such as inhibiting the reabsorption of water bymucosal surfaces, including airway and other surfaces.

The active compounds disclosed herein may be administered to mucosalsurfaces by any suitable means, including topically, orally, rectally,vaginally, ocularly and dermally, etc. For example, for the treatment ofconstipation, the active compounds may be administered orally orrectally to the gastrointestinal mucosal surface. The active compoundmay be combined with a pharmaceutically acceptable carrier in anysuitable form, such as sterile physiological or dilute saline or topicalsolution, as a droplet, tablet or the like for oral administration, as asuppository for rectal or genito-urethral administration, etc.Excipients may be included in the formulation to enhance the solubilityof the active compounds, as desired.

The active compounds disclosed herein may be administered to the airwaysurfaces of a patient by any suitable means, including as a spray, mist,or droplets of the active compounds in a pharmaceutically acceptablecarrier such as physiological or dilute saline solutions or distilledwater. For example, the active compounds may be prepared as formulationsand administered as described in U.S. Pat. No. 5,789,391 to Jacobus, thedisclosure of which is incorporated by reference herein in its entirety.

Solid or liquid particulate active agents prepared for practicing thepresent invention could, as noted above, include particles of respirableor non-respirable size; that is, for respirable particles, particles ofa size sufficiently small to pass through the mouth and larynx uponinhalation and into the bronchi and alveoli of the lungs, and fornon-respirable particles, particles sufficiently large to be retained inthe nasal airway passages rather than pass through the larynx and intothe bronchi and alveoli of the lungs. In general, particles ranging fromabout 1 to 5 microns in size (more particularly, less than about 4.7microns in size) are respirable. Particles of non-respirable size aregreater than about 5 microns in size, up to the size of visibledroplets. Thus, for nasal administration, a particle size in the rangeof 10-500 μm may be used to ensure retention in the nasal cavity.

In the manufacture of a formulation according to the invention, activeagents or the physiologically acceptable salts or free bases thereof aretypically admixed with, inter alia, an acceptable carrier. Of course,the carrier must be compatible with any other ingredients in theformulation and must not be deleterious to the patient. The carrier mustbe solid or liquid, or both, and is preferably formulated with thecompound as a unit-dose formulation, for example, a capsule, that maycontain 0.5% to 99% by weight of the active compound. One or more activecompounds may be incorporated in the formulations of the invention,which formulations may be prepared by any of the well-known techniquesof pharmacy consisting essentially of admixing the components.

Compositions containing respirable or non-respirable dry particles ofmicronized active agent may be prepared by grinding the dry active agentwith a mortar and pestle, and then passing the micronized compositionthrough a 400 mesh screen to break up or separate out largeagglomerates.

The particulate active agent composition may optionally contain adispersant which serves to facilitate the formulation of an aerosol. Asuitable dispersant is lactose, which may be blended with the activeagent in any suitable ratio (e.g., a 1 to 1 ratio by weight).

Active compounds disclosed herein may be administered to airway surfacesincluding the nasal passages, sinuses and lungs of a subject by ansuitable means know in the art, such as by nose drops, mists., etc. Inone embodiment of the invention, the active compounds of the presentinvention and administered by transbronchoscopic lavage. In a preferredembodiment of the invention, the active compounds of the presentinvention are deposited on lung airway surfaces by administering anaerosol suspension of respirable particles comprised of the activecompound, which the subject inhales. The respirable particles may beliquid or solid. Numerous inhalers for administering aerosol particlesto the lungs of a subject are known.

Inhalers such as those developed by Inhale Therapeutic Systems, PaloAlto, Calif., USA, may be employed, including but not limited to thosedisclosed in U.S. Pat. Nos. 5,740,794; 5,654,007; 5,458,135; 5,775,320;and 5,785,049, each of which is incorporated herein by reference. TheApplicant specifically intends that the disclosures of all patentreferences cited herein be incorporated by reference herein in theirentirety. Inhalers such as those developed by Dura Pharmaceuticals,Inc., San Diego, Calif., USA, may also be employed, including but notlimited to those disclosed in U.S. Pat. Nos. 5,622,166; 5,577,497;5,645,051; and 5,492,112, each of which is incorporated herein byreference. Additionally, inhalers such as those developed by AradigmCorp., Hayward, Calif., USA, may be employed, including but not limitedto those disclosed in U.S. Pat. Nos. 5,826,570; 5,813,397; 5,819,726;and 5,655,516, each of which is incorporated herein by reference. Theseapparatuses are particularly suitable as dry particle inhalers.

Aerosols of liquid particles comprising the active compound may beproduced by any suitable means, such as with a pressure-driven aerosolnebulizer or an ultrasonic nebulizer. See, e.g., U.S. Pat. No.4,501,729, which is incorporated herein by reference. Nebulizers arecommercially available devices which transform solutions or suspensionsof the active ingredient into a therapeutic aerosol mist either by meansof acceleration of compressed gas, typically air or oxygen, through anarrow venturi orifice or by means of ultrasonic agitation. Suitableformulations for use in nebulizers consist of the active ingredient in aliquid carrier, the active ingredient comprising up to 40% w/w of theformulation, but preferably less than 20% w/w. The carrier is typicallywater (and most preferably sterile, pyrogen-free water) or diluteaqueous alcoholic solution. Perfluorocarbon carriers may also be used.Optional additives include preservatives if the formulation is not madesterile, for example, methyl hydroxybenzoate, antioxidants, flavoringagents, volatile oils, buffering agents and surfactants.

Aerosols of solid particles comprising the active compound may likewisebe produced with any solid particulate medicament aerosol generator.Aerosol generators for administering solid particulate medicaments to asubject produce particles which are respirable, as explained above, andgenerate a volume of aerosol containing predetermined metered dose ofmedicament at a rate suitable for human administration. One illustrativetype of solid particulate aerosol generator is an insufflator. Suitableformulations for administration by insufflation include finelycomminuted powders which may be delivered by means of an insufflator ortaken into the nasal cavity in the manner of a snuff. In theinsufflator, the powder (e.g., a metered dose thereof effective to carryout the treatments described herein) is contained in capsules orcartridges, typically made of gelatin or plastic, which are eitherpierced or opened in situ and the powder delivered by air drawn throughthe device upon inhalation or by means of a manually-operated pump. Thepowder employed in the insufflator consists either solely of the activeingredient or of powder blend comprising the active ingredient, asuitable powder diluent, such as lactose, and an optional surfactant.The active ingredient typically comprises of 0.1 to 100% w/w of theformulation. A second type of illustrative aerosol generator comprises ametered dose inhaler. Metered dose inhalers are pressurized aerosoldispensers, typically containing a suspension or solution formulation ofactive ingredient in a liquified propellant. During use, these devicesdischarge the formulation through a valve adapted to deliver a meteredvolume, typically from 10 to 150 μl, to produce a fine particle spraycontaining the active ingredient. Suitable propellants include certainchlorofluorocarbon compounds, for example, dichlorodifluoromethane,trichlorofluoromethane, dichlorotetrafluoroethane and mixtures thereof.The formulation may additionally contain one of more co-solvents, forexample, ethanol, surfactants, such as oleic acid or sorbitan trioleate,antioxidants and suitable flavoring agents.

The aerosol, whether formed from solid or liquid particles, may beproduced by the aerosol generator at a rate of from about 10 to 150liters per minute, more preferable from 30 to 150 liters per minute, andmost preferably about 60 liters per minute. Aerosols containing greateramounts of medicament may be administered more rapidly.

The dosage of the active compounds disclosed herein will vary dependingon the condition being treated and the state of the subject, butgenerally may be from about 0.01, 0.03, 0.05, 0.1 to 1, 5, 10 or 20 mgof the pharmaceutic agent, deposited on the airway surfaces. The dailydose may be divided among one or multiple unit dose administrations. Thegoal is to achieve a concentration of the pharmaceutic agents on lungairway surfaces of between 10⁻⁹-10⁴ M.

In another embodiment, they are administered by administering an aerosolsuspension of respirable or non-respirable particles (preferablynon-respirable particles) comprised of active compound, which thesubject inhales through the nose. The respirable or non-respirableparticles may be liquid or solid. The quantity of active agent includedmay be an amount of sufficient to achieve dissolved concentrations ofactive agent on the airway surfaces of the subject of from about 10⁻⁹,10⁻⁸, or 10⁻⁷ to about 10⁻³, 10⁻², 10⁻¹ moles/liter, and more preferablyfrom about 10⁻⁹ to about 10⁻⁴ moles/liter.

The dosage of active compound will vary depending on the condition beingtreated and the state of the subject, but generally may be an amountsufficient to achieve dissolved concentrations of active compound on thenasal airway surfaces of the subject from about 10⁻⁹, 10⁻⁸, 10⁻⁷ toabout 10⁻³, 10⁻², or 10⁻¹ moles/liter, and more preferably from about10⁻⁷ to about 10⁻⁴ moles/liter. Depending upon the solubility of theparticular formulation of active compound administered, the daily dosemay be divided among one or several unit dose administrations. The dailydose by weight may range from about 0.01, 0.03, 0.1, 0.5 or 1.0 to 10 or20 milligrams of active agent particles for a human subject, dependingupon the age and condition of the subject. A currently preferred unitdose is about 0.5 milligrams of active agent given at a regimen of 2-10administrations per day. The dosage may be provided as a prepackagedunit by any suitable means (e.g., encapsulating a gelatin capsule).

In one embodiment of the invention, the particulate active agentcomposition may contain both a free base of active agent and apharmaceutically acceptable salt to provide both early release andsustained release of active agent for dissolution into the mucussecretions of the nose. Such a composition serves to provide both earlyrelief to the patient, and sustained relief over time. Sustained relief,by decreasing the number of daily administrations required, is expectedto increase patient compliance with the course of active agenttreatments.

Pharmaceutical formulations suitable for airway administration includeformulations of solutions, emulsions, suspensions and extracts. Seegenerally, J. Nairn, Solutions, Emulsions, Suspensions and Extracts, inRemington: The Science and Practice of Pharmacy, chap. 86 (19^(th) ed.1995), incorporated herein by reference. Pharmaceutical formulationssuitable for nasal administration may be prepared as described in U.S.Pat. No. 4,389,393 to Schor; U.S. Pat. No. 5,707,644 to Illum; U.S. Pat.No. 4,294,829 to Suzuki; and U.S. Pat. No. 4,835,142 to Suzuki, thedisclosures of which are incorporated by reference herein in theirentirety.

Mists or aerosols of liquid particles comprising the active compound maybe produced by any suitable means, such as by a simple nasal spray withthe active agent in an aqueous pharmaceutically acceptable carrier, suchas a sterile saline solution or sterile water. Administration may bewith a pressure-driven aerosol nebulizer or an ultrasonic nebulizer. Seee.g. U.S. Pat. Nos. 4,501,729 and 5,656,256, both of which areincorporated herein by reference. Suitable formulations for use in anasal droplet or spray bottle or in nebulizers consist of the activeingredient in a liquid carrier, the active ingredient comprising up to40% w/w of the formulation, but preferably less than 20% w/w. Typicallythe carrier is water (and most preferably sterile, pyrogen-free water)or dilute aqueous alcoholic solution, preferably made in a 0.12% to 0.8%solution of sodium chloride. Optional additives include preservatives ifthe formulation is not made sterile, for example, methylhydroxybenzoate, antioxidants, flavoring agents, volatile oils,buffering agents, osmotically active agents (e.g. mannitol, xylitol,erythritol) and surfactants.

Compositions containing respirable or non-respirable dry particles ofmicronized active agent may be prepared by grinding the dry active agentwith a mortar and pestle, and then passing the micronized compositionthrough a 400 mesh screen to break up or separate out largeagglomerates.

The particulate composition may optionally contain a dispersant whichserves to facilitate the formation of an aerosol. A suitable dispersantis lactose, which may be blended with the active agent in any suitableratio (e.g., a 1 to 1 ratio by weight).

The compounds of formula (I) may be synthesized according to proceduresknown in the art. A representative synthetic procedure is shown in thescheme below:

These procedures are described in, for example, E. J. Cragoe, “TheSynthesis of Amiloride and Its Analogs” (Chapter 3) in Amiloride and ItsAnalogs, pp. 25-36, incorporated herein by reference. Other methods ofpreparing the compounds are described in, for example, U.S. Pat. No.3,313,813, incorporated herein by reference. See in particular MethodsA, B, C, and D described in U.S. Pat. No. 3,313,813. Other methodsuseful for the preparation of these compounds, especially for thepreparation of the novel HNR³R⁴ fragment. are described in, for example233377US(Ser. No. 60/367,497), 213706US(Ser. No. 10/076,571),241739US-PROV(Ser. No. 60/495,725), 241741US-PROV(Ser. No. 60/495,712)and 241740US-PROV(Ser. No. 60/495,720), incorporated herein byreference. Several assays may be used to characterize the compounds ofthe present invention. Representative assays are discussed below.

In Vitro Measure of Sodium Channel Blocking Activity and Reversibility

One assay used to assess mechanism of action and/or potency of thecompounds of the present invention involves the determination of lumenaldrug inhibition of airway epithelial sodium currents measured undershort circuit current (I_(SC)) using airway epithelial monolayersmounted in Ussing chambers. Cells obtained from freshly excised human,dog or sheep airways are seeded onto porous 0.4 micron Snapwell™ Inserts(CoStar), cultured at air-liquid interface (ALI) conditions inhormonally defined media, and assayed for sodium transport activity(I_(SC) in μA/cm²) while bathed in Krebs Bicarbonate Ringer (KBR) inTissing chambers. All test drug additions are to the lumenal bath withhalf-log dose addition protocols (from 1×10⁻¹¹ M to 3×10⁻⁵ M), and thecumulative change in I_(SC) (inhibition) recorded. All drugs areprepared in dimethyl sulfoxide as stock solutions at a concentration of1×10⁻² M and stored at −20° C. Six preparations are typically run inparallel; one preparation per run incorporates a positive control. Alldata from the voltage clamps are collected via a computer interface andanalyzed off-line.

Dose-effect relationships for all compounds are considered and analyzedby the Prism 3.0 program. EC₅₀ values, maximal effective concentrationsare calculated and compared to positive controls.

In Vitro Durability of Sodium Channel Blockers: Surface LiquidAbsorption, Transport, and Metabolic Profile

The airway bronchial epithelium is an absorptive epithelium (activelyabsorbs sodium and therefore water from the lumenal to serosaldirection. Using a gravimetric (weighing) procedure, the lumenal surfaceliquid is weighed and changes recorded up to 36 h. An applied startingvolume of buffer (modified Krebs-Henseleit Bicarbonate buffer solution)with and without equimolar concentrations of selected novel orcommercially available sodium channel blockers are added to the startingbuffer, and at selected time points the lumenal surface liquid mass isweighed and the mass recorded in mg. In addition, during the assay,samples are collected from both the surface liquid and serosalcompartment, after which the wells re-weighted and weights recorded. Thesamples collected are analyzed using HPLC and or mass spectrometry, andthe concentration of sodium channel blocker calculated, with anyconjugate or metabolite noted.

Solubility of Compounds in Water or Sodium Chloride Solution

Compound solubility was measured in water, 0.12 or 0.9% sodium chloridesolution at ambient temperature for up to 10 days. Using a UV/VisableSpectrophotometer and applying Beer's Law with the calculated extinctioncoeffecient of amiloride (18.6 mM, absorbance values at 362 nm takenfrom D. Mazzo 1986) the free base concentration in solution wascalculated at specified time points. All samples were stored for theduration of the experiment in a single/closure system consisting ofglass vials with a stopper-top closure. The vials were maintained atambient temperature, in the dark, and in the upright position. Compoundstability was measured using reverse phase high performance liquidchromatography on the final filtered pulled sample (day 10).

Confocal Microscopy Assay of Amiloride Congener Uptake

Virtually all molecules studied fluoresce in the ultraviolet range. Thisproperty of these molecules may be used to directly measure cellularupdate using x-z confocal microscopy. Equimolar concentrations ofexperimental compounds and positive controls including amiloride andcompounds that demonstrate rapid uptake into the cellular compartment(benzamil and phenamil) are placed on the apical surface of airwaycultures on the stage of the confocal microscope. Serial x-z images areobtained with time and the magnitude of fluorescence accumulating in thecellular compartment is quantitated and plotted as a change influorescence versus time.

Pharmacological Effects and Mechanism of Action of the Drug in Animals

The effect of compounds for enhancing mucociliary clearance (MCC) can bemeasured using an in vivo model described by Sabater et al., Journal ofApplied Physiology, 1999, pp. 2191-2196, incorporated herein byreference.

In Vivo Assay in Sheep

Methods

Animal Preparation: Adult ewes (ranging in weight from 25 to 35 kg) wererestrained in an upright position in a specialized body harness adaptedto a modified shopping cart. The animals' heads were immobilized andlocal anesthesia of the nasal passage was induced with 2% lidocaine. Theanimals were then nasally intubated with a 7.5 mm internal diameterendotracheal tube (ETT). The cuff of the ETT was placed just below thevocal cords and its position was verified with a flexible bronchoscope.After intubation the animals were allowed to equilibrate forapproximately 20 minutes prior to initiating measurements of mucociliaryclearance.

Administration of Radio-aerosol: Aerosols of ^(99m)Tc-Human serumalbumin (3.1 mg/ml; containing approximately 20 mCi) were generatedusing a Raindrop Nebulizer which produces a droplet with a medianaerodynamic diameter of 3.6 μm. The nebulizer was connected to adosimetry system consisting of a solenoid valve and a source ofcompressed air (20 psi). The output of the nebulizer was directed into aplastic T connector; one end of which was connected to the endotrachealtube, the other was connected to a piston respirator. The system wasactivated for one second at the onset of the respirator's inspiratorycycle. The respirator was set at a tidal volume of 500 mL, aninspiratory to expiratory ratio of 1:1, and at a rate of 20 breaths perminute to maximize the central airway deposition. The sheep breathed theradio-labeled aerosol for 5 minutes. A gamma camera was used to measurethe clearance of ^(99m)Tc-Human serum albumin from the airways. Thecamera was positioned above the animal's back with the sheep in anatural upright position supported in a cart so that the field of imagewas perpendicular to the animal's spinal cord. External radio-labeledmarkers were placed on the sheep to ensure proper alignment under thegamma camera. All images were stored in a computer integrated with thegamma camera. A region of interest was traced over the imagecorresponding to the right lung of the sheep and the counts wererecorded. The counts were corrected for decay and expressed aspercentage of radioactivity present in the initial baseline image. Theleft lung was excluded from the analysis because its outlines aresuperimposed over the stomach and counts can be swallowed and enter thestomach as radio-labeled mucus.

Treatment Protocol (Assessment of activity at t-zero): A baselinedeposition image was obtained immediately after radio-aerosoladministration. At time zero, after acquisition of the baseline image,vehicle control (distilled water), positive control (amiloride), orexperimental compounds were aerosolized from a 4 ml volume using a PariLC JetPlus nebulizer to free-breathing animals. The nebulizer was drivenby compressed air with a flow of 8 liters per minute. The time todeliver the solution was 10 to 12 minutes. Animals were extubatedimmediately following delivery of the total dose in order to preventfalse elevations in counts caused by aspiration of excess radio-tracerfrom the ETT. Serial images of the lung were obtained at 15-minuteintervals during the first 2 hours after dosing and hourly for the next6 hours after dosing for a total observation period of 8 hours. Awashout period of at least 7 days separated dosing sessions withdifferent experimental agents.

Treatment Protocol (Assessment of Activity at t-4 hours): The followingvariation of the standard protocol was used to assess the durability ofresponse following a single exposure to vehicle control (distilledwater), positive control compounds (amiloride or benzamil), orinvestigational agents. At time zero, vehicle control (distilled water),positive control (amiloride), or investigational compounds wereaerosolized from a 4 ml volume using a Pari LC JetPlus nebulizer tofree-breathing animals. The nebulizer was driven by compressed air witha flow of 8 liters per minute. The time to deliver the solution was 10to 12 minutes. Animals were restrained in an upright position in aspecialized body harness for 4 hours. At the end of the 4-hour periodanimals received a single dose of aerosolized ^(99m)Tc-Human serumalbumin (3.1 mg/ml; containing approximately 20 mCi) from a RaindropNebulizer. Animals were extubated immediately following delivery of thetotal dose of radio-tracer. A baseline deposition image was obtainedimmediately after radio-aerosol administration. Serial images of thelung were obtained at 15-minute intervals during the first 2 hours afteradministration of the radio-tracer (representing hours 4 through 6 afterdrug administration) and hourly for the next 2 hours after dosing for atotal observation period of 4 hours. A washout period of at least 7 daysseparated dosing sessions with different experimental agents.

Statistics: Data were analyzed using SYSTAT for Windows, version 5. Datawere analyzed using a two-way repeated ANOVA (to assess overalleffects), followed by a paried t-test to identify differences betweenspecific pairs. Significance was accepted when P was less than or equalto 0.05. Slope values (calculated from data collected during the initial45 minutes after dosing in the t-zero assessment) for mean MCC curveswere calculated using linear least square regression to assessdifferences in the initial rates during the rapid clearance phase.

Examples

Having generally described this invention, a further understanding canbe obtained by reference to certain specific examples which are providedherein for purposes of illustration only and are not intended to belimiting unless otherwise specified.

Preparation of Sodium Channel Blockers

Materials and methods. All reagents and solvents were purchased fromAldrich Chemical Corp. and used without further purification. NMRspectra were obtained on either a Bruker WM 360 (¹H NMR at 360 MHz and¹³C NMR at 90 MHz) or a Bruker AC 300 (¹H NMR at 300 MHz and ¹³C NMR at75 MHz). Flash chromatography was performed on a Flash Elute™ systemfrom Elution Solution (PO Box 5147, Charlottesville, Va. 22905) chargedwith a 90 g silica gel cartridge (40M FSO-0110-040155, 32-63 μm) at 20psi (N2). GC-analysis was performed on a Shimadzu GC-17 equipped with aHeliflex Capillary Column (Alltech); Phase: AT-1, Length: 10 meters, ID:0.53 mm, Film: 0.25 micrometers. GC Parameters: Injector at 320° C.,Detector at 320° C., FID gas flow: H₂ at 40 ml/min, Air at 400 ml/min.Carrier gas: Split Ratio 16:1, N₂ flow at 15 ml/min., N₂ velocity at 18cm/sec. The temperature program is 70° C. for 0-3 min, 70-300° C. from3-10 min, 300° C. from 10-15 min.

HPLC analysis was performed on a Gilson 322 Pump, detector UV/Vis-156 at360 nm, equipped with a Microsorb MV C8 column, 100 A, 25 cm. Mobilephase: A=acetonitrile with 0.1% TFA, B=water with 0.1% TFA. Gradientprogram: 95:5 B:A for 1 min, then to 20:80 B:A over 7 min, then to 100%A over 1 min, followed by washout with 100% A for 11 min, flow rate: 1ml/min.

Example 1 Synthesis of2-(4-{4-[N′-(3,5-diamino-6-chloropyrazine-2-carbonyl)guanidino]-butyl}phenoxy)-N-(3-dimethylaminopropyl)acetamidedimethanesulfonate (PSA 24304)

(4-{4-[(3-Dimethylaminopropylcarbamoyl)methoxy]phenyl}butyl)carbamicacid benzyl ester (3)

A solution of 1(0.50 g, 1.39 mmol) and CDI (0.25 g, 1.54 mmol) in THF(15 mL) was heated at 40° C. for 1 h. Then 2 (0.15 g, 1.47 mmol) wasadded into the reaction mixture at that temperature. The resultingsolution was slowly cooled down to room temperature and further stirredat the temperature overnight. After that, the solvent was removed underreduced pressure and the residue was purified by Flash™ chromatography(BIOTAGE, Inc) (9:0.9:0.1 dichloromethane/methanol/concentrated ammoniumhydroxide, v/v) to provide 3 (0.4 g, 61%) as a white solid. ¹H NMR (500MHz, CD₃OD) δ 1.48 (m, 211), 1.64 (m, 2H), 1.72 (m, 2H), 2.14 (s, 6H),2.27 (m, 2H), 2.56 (m, 2H), 3.10 (m, 2H), 3.29 (m, 3H), 4.41 (s, 2H),5.09 (s, 2H), 6.85 (d, 2H), 7.09 (d, 2H), 7.31 (m, 5H). m/z (ESI) 442.

2-[4-(4-Aminobutyl)phenoxy]-N-(3-dimethylaminopropyl)acetamide (4)

A suspension of 3 (377 mg, 0.85 mmol) and 10% palladium on carbon (0.30g, 50% wet) in methanol (15 mL) was stirred at room temperature for 2 hunder atmospheric pressure of hydrogen. The mixture was then filteredthrough a Celite pad and the solvent was evaporated to provide 4 (208mg, 80%) as a white solid. The crude product was directly used in thenext step without purification. 1H NMR (300 MHz, CD₃OD) δ 1.61 (m, 6H),2.11 (s, 6H), 2.32 (m, 2H), 2.65 (m, 4H), 3.28 (m, 2H), 4.45 (s, 2H),6.90 (d, 2H), 7.19 (d, 2H). m/z (ESI) 308.

2-(4-{4-[N′-(3,5-Diamino-6-chloropyrazine-2-carbonyl)guanidino]butyl}phenoxy)-N-(3-dimethylaminopropyl)acetamide(6)

1-(3,5-Diamino-6-chloropyrazine-2-carbony)-2-methylisothioureahydriodide (292 mg, 0.75 mmol) 5 was added to a solution of compound 4(200 mg, 0.65 mmol), DIPEA (0.39 mL, 2.25 mmol), and ethanol (5 mL). Thereaction mixture was stirred at 65° C. for 3 h. The solvent was removedunder reduced pressure and the residue was purified by preparative TLC(80:18:2 dichloromethane/methanol/concentrated ammonium hydroxide, v/v)to provide 6 (190 mg, 56%) as a light yellow solid. ¹H NMR (300 MHz,CD₃OD) δ 1.65 (m, 6H), 2.15 (s, 6H), 2.32 (m, 2H), 2.64 (m, 2H), 3.27(m, 8H), 4.49 (s, 2H), 6.89 (d, 2H), 7.15 (d, 2H). m/z (ESI) 520.

Methanesulfonic acid (67.2 mg, 0.70 mmol) was added to the solution of 6(182 mg, 0.35 mmol) in ethanol (4 mL). The resulting solution wasstirred at room temperature for 0.5 h; then the solvent was completelyevaporated, affording 212 mg (85%) of 7 as a light yellow solid. m.p.187-190° C. ¹H NMR (300 MHz, CD₃OD) δ 1.65 (m, 4H), 1.90 (m, 2H), 2.67(m, 8H), 2.85 (s, 6H), 3.15 (m, 2H), 3.40 (m, 4H), 4.49 (s, 2H), 6.89(d, 2H), 7.15 (d, 2H). m/z (APCI) 520 [C₂₃H₃₄ClN₉O₃+H]⁺.

Example 2 Synthesis and Physical Properties of Selected Soluble Amides

Utilizing the procedures exemplified in Example 1 and Scheme 1, thecompounds listed in Table 1 were prepared.

TABLE 1 Physical Properties of Selected Amides

Molecular HPLC² Formula Molecular Melting Analysis PSAI# R = •2CH₃SO₃HWeight Point ° C. NMR¹ (%) M/Z³ 23778 —NH(CH₂)₂NH2 C₂₀H₂₈ClN₉O₃ 670.16  105-107° (d) Consistent 95.4 478 23185a —NH(CH₂)₂N(CH₃)₂ C22H32ClN₉O₃698.22 97-99° Consistent 97.4 506 24304 NH(CH₂)₃N(CH₃)₂ C₂₃H₃₄ClN₉O₃712.25 187-190° Consistent 95.8 520 24305 NH(CH₂)₄N(CH₃)₂ C₂₄H₃₆ClN₉O₃726.27 188-190° Consistent 97.0 534 23450 NH(CH₂)₂N(CH₂CH₂OH)₂C₂₄H₃₆ClN₉O₅ 758.27 87-89° Consistent 97.7 566 19913

C₂₂H₃₀ClN₉O₃ 696.21 72-74° Consistent 97.9 504 Notes: 1. NMR = 500 MHz¹H NMR Spectrum (CD₃OD). 2. HPLC—Polarity dC18 Column, Detector @ 200nM. 3. M/Z = APCI Mass Spectrum [Free Base + H]⁺.

Example 3 Solubility of Selected Amides

Table 2 gives the solubility in saline of selected amide bis methanesulfonic acid salts and compares them to the mono addition methanesulfonic acid salt of PSA 9714.

TABLE 2 Solubility of Selected Amides

PSA# R = S¹ S²  9714 —NH₂ <0.1 mg/ml   <0.1 mg/ml   237778—NH(CH₂)₂NH₂ >5 mg/ml  23185a —NH(CH₂)₂N(CH₃)₂ >5 mg/ml  24304—NH(CH₂)₃N(CH₃)₂ >5 mg/ml  24305 —NH(CH₂)₄N(CH₃)₂ >5 mg/ml >5 mg/ml 23450 NH(CH₂)₂N(CH₂CH₂OH)₂ >5 mg/ml >5 mg/ml  19913

>5 mg/ml S¹ = Solubility in 0.12% NaCl Solution S² = Solubility in 0.9%NaCl Solution (normal Saline)

Example 4 Sodium Channel Blocking Activity of Selected Soluble Amides

Utilizing the tests set forth above, Table 3 summaries the ENaC blockingability of some of the amides of this invention when assayed in caninebronchial epithelium.

TABLE 3 Epithelial Sodium Channel Blocking Activity of Selected Amides

IC₅₀ Fold Amiloride** PSA# R = (nM) (PSA4022 = 100)  9714 —NH₂ 15.2 +6.3  62  (n = 36) 237778 —NH(CH₂)₂NH₂ 5 ± 2 146 (n = 4)  23185a—NH(CH₂)₂N(CH₃)₂ 14 ± 9  59 (n = 3)  24304 —NH(CH₂)₃N(CH₃)₂ 4 ± 2 173 (n= 7)  24305 —NH(CH₂)₄N(CH₃)₂ 6 ± 5 152 (n = 6)  23450NH(CH₂)₂N(CH₂CH₂OH)₂ 18 38  19913

9 ± 1 (n = 2) 98 **Relative potency for PSA4022 = 100 using IC₅₀ fromPSA4022 in same run.

Example 5N-(3,5-Diamino-6-chloropyrazine-2-carbonyl)-N′-{4-[4-(piperazine-1-carbonyl)phenyl]butyl}guanidinebis-methanesulfonate (PSA23607) 4-(4-Aminobutyl)benzoic acid (8)

A solution of sodium hydroxide (0.69 g, 17.37 mmol) in water (30 mL) wasadded to a solution of 24 (1.2 g, 5.79 mmol) in methanol (30 mL) andstirred at room temperature for 48 h. Then the solvent was removed underreduced pressure. Water (20 mL) was added and pH was adjusted to 7 withHCl. The white solid precipitate was filtered off, washed with water anddried in vacuum. The crude product 8 (1.39 g) was obtained as a whitesolid and used for the next step without further purification.

4-(4-Benzyloxycarbonylaminobutyl)benzoic acid (9)

Sodium hydrogencarbonate (0.95 g, 11.32 mmol) was added into asuspension of 30 in THF (120 mL), followed by water (10 mL), affording aclear solution. Benzyl chloroformate (1.21 mL, 8.49 mmol) was then addedinto the reaction mixture at 0° C. The reaction mixture was then stirredat room temperature overnight. After that, the solvent was removed underreduced pressure. Ethyl acetate (70 mL) was added to the residue and thesolution was washed with 2N HCl (2×30 mL) and water (2×50 mL), thendried in vacuum. 1.82 g (98%) of 9 was obtained as a white solid. ¹H NMR(300 MHz, DMSO-d₆) δ 1.11 (m, 2H), 1.28 (m, 2H), 2.33 (m, 2H), 3.02 (m,2H), 5.01 (m, 2H), 7.15 (m, 7H), 7.93 (d 2H).

N-(3,5-Diamino-6-chloropyrazine-2-carbonyl)-N′-{4-[4-(piperazine-1-carbonyl)phenyl]butyl}guanidinebis-methanesulfonate (PSA23607)

Compound 9 was converted to PSA236507 utilizing the general methodsdescribed in Example 1 yielding the desired product, melting poing122-124° C., 500 MHz ¹H NMR Spectrum (CD₃OD) was consistent withassigned structure, HPLC Analysis 98.5% (area percent), Polarity dC18Column, Detector @ 220 nm, ESI mass Spectrum m/z 474 [C₂₁H₂₈ClN₉O₂+H]⁺.PSA23607 had an IC₅₀ of 12.5±0.5 nM on canine bronchial epithelial andhad a solubility of greater than 5 mg/ml in 0.12% saline solution.

Example 63-[4-(4-Aminobutyl)phenoxy]-2-tert-butoxycarbonylaminopropionic acidmethyl ester (13)3-[4-(4-Benzyloxycarbonylaminobutyl)phenoxy]-2-(tritylamino)propionicacid methyl ester (10)

Commercially available N-trityl-L-serine methyl ester (1.60 g, 5.34mmol) was combined with triphenylphosphine (1.28 g, 4.88 mmol) and[4-(4-hydroxyphenyl)butyl]carbamic acid benzyl ester (2.0 g, 6.68 mmol)in benzene (40 mL) at room temperature. Diisopropyl azodicarboxylate(0.958 mL, 4.86 mmol) was added dropwise and the reaction was stirredfor 14 days. The solvent was removed under reduced pressure and theresidue was purified by column chromatography (silica gel, eluent: 6:1,v/v dichloromethane/ethyl acetate) to provide compound 10 (2.22 g, 51%).¹H NMR (300 MHz, CDCl₃) δ 7.52 (m, 6H), 7.39-7.14 (m, 14H), 7.06 (d,2H), 6.79 (d, 2H), 5.09(s, 2H), 4.72 (m, 1H), 4.24 (m, 1H), 4.01 (m,1H), 3.72 (m, 1H), 3.22 (s, 3H), 3.18 (m, 2H), 2.88 (d, 1H), 2.57 (m,2H), 1.66-1.48 (m, 4H). R_(f)=0.91 (5:1 v/v dichloromethane/ethylacetate).

3-[4-(4-Benzyloxycarbonylaminobutyl)phenoxy]-2-tert-butoxycarbonylamino-propionicacid methyl ester (12)

Compound 10 (2.22 g, 3.45 mmol) was dissolved in a mixture ofdichloromethane/water (25 mL/0.5 mL) then trifluoroacetic acid (0.75 mL,10.0 mmol) was added and the reaction was stirred for 2 h. The solventwas removed under reduced pressure and the residue was dissolved indichloromethane (25 mL) and treated with triethylamine (0.72 mL, 5.12mmol) and di-tert-butyl dicarbonate (0.829 g, 3.79 mmol) for 72 h.Removal of the solvents under reduced pressure followed by columnchromatography (silica gel, eluent: 9:1, v/v dichloromethane/ethylacetate) provided compound 12 (0.90 g, 52%). ¹H NMR (300 MHz, CDCl₃) δ7.34 (m, 5H), 7.07 (d, 2H), 6.79 (d, 2H), 5.50 (d, 1H), 5.10 (s, 2H),4.68 (m, 2H), 4.38 (m, 1H), 4.17 (m, 1H), 3.77 (s, 3H), 3.20 (m, 2H),2.57 (m, 2H), 1.67-1.48 (m, 4H), 1.45 (s, 9H). m/z (APCI) 401[C₂₇H₃₆N₂O₇−Boc+H]⁺.

3-[4-(4-Aminobutyl)phenoxy]-2-tert-butoxycarbonylaminopropionic acidmethyl ester (13)

Compound 12 (505 mg, 1.00 mmol) was dissolved in methanol (20 mL) and10% palladium on carbon (100 mg) was added. The flask was evacuated,filled with hydrogen gas under balloon pressure and stirred overnight.Filtration through celite to remove the catalyst followed by removal ofthe solvent under reduced pressure provided compound 13 (366 mg, 98%).¹H NMR (300 MHz, CDCl₃) δ 7.08 (d, 2H), 6.80 (d, 2H), 5.51 (d, 1H), 4.66(m, 1H), 4.38 (m, 1H), 4.17 (m, 1H), 3.78 (s, 3H), 2.73 (m, 2H), 2.58(m, 2H), 1.90 (br s, 2H), 1.62 (m, 2H), 1.50 (m, 2H), 1.48 (s, 9H).

Example 73-[4-(4-Aminobutyl)phenoxy]-2-tert-butoxycarbonylaminopropionic acidmethyl ester (16)

3-[4-(4-Benzyloxycarbonylaminobutyl)phenoxy]-2-(tritylamino)propionicacid methyl ester (14)

Commercially available N-trityl-L-serine methyl ester (1.60 g, 5.34mmol) was combined with triphenylphosphine (1.28 g, 4.88 mmol) and[4-(4-hydroxyphenyl)butyl]carbamic acid benzyl ester (2.0 g, 6.68 mmol)in benzene (40 mL) at room temperature. Diisopropyl azodicarboxylate(0.958 mL, 4.86 mmol) was added dropwise and the reaction was stirredfor 14 days. The solvent was removed under reduced pressure and theresidue was purified by column chromatography (silica gel, eluent: 6:1,v/v dichloromethane/ethyl acetate) to provide compound 141 (2.22 g,51%). ¹H NMR (300 MHz, CDCl₃) δ 7.52 (m, 6H), 7.39-7.14 (m, 14H), 7.06(d, 2H), 6.79 (d, 2H), 5.09(s, 2H), 4.72 (m, 1H), 4.24 (m, 1H), 4.01 (m,1H), 3.72 (m, 1H), 3.22 (s, 3H), 3.18 (m, 2H), 2.88 (d, 1H), 2.57 (m,2H), 1.66-1.48 (m, 4H). R_(f)=0.91 (5:1 v/v dichloromethane/ethylacetate).

3-[4-(4-Benzyloxycarbonylaminobutyl)phenoxy]-2-tert-butoxycarbonylamino-propionicacid methyl ester (15)

Compound 14 (2.22 g, 3.45 mmol) was dissolved in a mixture ofdichloromethane/water (25 mL/0.5 mL) then trifluoroacetic acid (0.75 mL,10.0 mmol) was added and the reaction was stirred for 2 h. The solventwas removed under reduced pressure and the residue was dissolved indichloromethane (25 mL) and treated with triethylamine (0.72 mL, 5.12mmol) and di-tert-butyl dicarbonate (0.829 g, 3.79 mmol) for 72 h.Removal of the solvents under reduced pressure followed by columnchromatography (silica gel, eluent: 9:1, v/v dichloromethane/ethylacetate) provided compound 15 (0.90 g, 52%). ¹H NMR (300 MHz, CDCl₃) δ7.34 (m, 5H), 7.07 (d, 2H), 6.79 (d, 2H), 5.50 (d, 1H), 5.10 (s, 2H),4.68 (m, 2H), 4.38 (m, 1H), 4.17 (m, 1H), 3.77 (s, 3H), 3.20 (m, 2H),2.57 (m, 2H), 1.67-1.48 (m, 4H), 1.45 (s, 9H). m/z (APCI) 401[C₂₇H₃₆N₂O₇−Boc+H]⁺.

3-[4-(4-Aminobutyl)phenoxy]-2-tert-butoxycarbonylaminopropionic acidmethyl ester (16)

Compound 15 (505 mg, 1.00 mmol) was dissolved in methanol (20 mL) and10% palladium on carbon (100 mg) was added. The flask was evacuated,filled with hydrogen gas under balloon pressure and stirred overnight.Filtration through celite to remove the catalyst followed by removal ofthe solvent under reduced pressure provided compound 16 (366 mg, 98%).¹H NMR (300 MHz, CDCl₃) δ 7.08 (d, 2H), 6.80 (d, 2H), 5.51 (d, 1H), 4.66(m, 1H), 4.38 (m, 1H), 4.17 (m, 1H), 3.78 (s, 3H), 2.73 (m, 2H), 2.58(m, 2H), 1.90 (br s, 2H), 1.62 (m, 2H), 1.50 (m, 2H), 1.48 (s, 9H).

Example 8{4-[4-(3-tert-Butoxycarbonylamino-3-carbamoylpropoxy)phenyl]butyl}carbamicacid benzyl ester (20)

2-Amino-4-hydroxybutyric acid methyl ester hydrochloride (17)

A suspension of DL-homoserine (1.00 g, 8.39 mmol) in methanol (40 mL)was placed in an ice bath. Trimethylsilyl chloride (2.34 mL, 18.5 mmol)was added dropwise via syringe. The reaction mixture gradually becamehomogenous and was further stirred at rt for 14 h, concentrated byrotary evaporation, and further dried under high vacuum. The crude oilthus obtained was used for the next step without further purification.¹H NMR (300 MHz, CD₃OD) δ 2.00-2.24 (m, 2H), 3.70-3.80 (m, 2H), 3.85 (s,3H), 4.12-4.22 (m, 1H). m/z (ESI) 134 [C₅H₁₁NO₃+H]⁺.

2-tert-Butoxycarbonylamino-4-hydroxybutyric acid methyl ester (18)

2-Amino-4-hydroxybutyric acid methyl ester hydrochloride (17) wassuspended in anhydrous THF (15 mL) and placed in an ice bath.Diisopropylethylamine (2.92 mL, 16.8 mmol) was added via syringe,followed by the addition of DMAP (205 mg, 1.68 mmol) and Boc₂O (3.85 g,17.6 mmol). The mixture was stirred at 0° C. for 10 min and at roomtemperature for 14 h. Solvent was removed under reduced pressure andresidue was taken up by ethyl acetate (100 mL), washed with water (30mL×2) and brine (40 mL), dried over sodium sulfate, and concentrated.The colorless oil (1.96 g) was used for the next step without furtherpurification. ¹H NMR (300 MHz, CDCl₃) δ 1.45 (s, 9H), 2.05-2.28 (m, 2H),3.68-3.75 (m, 2H), 3.80 (s, 3H), 4.08-4.15 (m, 1H), 5.30-5.41 (m, 1H).m/z (ESI) 234 [C₁₀H₁₉NO₅+H]⁺.

4-Bromo-2-tert-butoxycarbonylaminobutyric acid methyl ester (19)

A solution of triphenylphosphine (2.20 g, 8.39 mmol) in dry CH₂Cl₂ (20mL) was added dropwise via syringe to a solution of N-Boc homoserinemethyl ester 18 (8.39 mmol) and carbon tetrabromide (4.18 g, 12.60 mmol)in dry CH₂Cl₂ (20 mL). The resulting dark solution was stirred at roomtemperature for 16 h. Hexanes was added and precipitates were removed bysuction filtration. The filtrate was concentrated under reduced pressureand subject to flash silica gel column chromatography using ethylacetate/hexanes (1:10, v/v then 1:6, v/v) to give the desired product 19as a yellow oil (501 mg, 20% overall yield from homoserine). ¹H NMR (300MHz, CDCl₃) δ 1.45 (s, 9H), 2.12-2.48 (m, 2H), 3.39-3.47 (m, 2H), 3.78(s, 3H), 4.33-4.48 (m, 1H), 5.10-5.22 (m, 1H). m/z (ESI) 296[C₁₀H₁₈BrNO₄+H]⁺.

4-[4-(4-Benzyloxycarbonylaminobutyl)phenoxy]-2-tert-butoxycarbonylamino-butyricacid methyl ester (20)

Potassium carbonate (935 mg, 6.77 mmol) was added in one portion to asolution of 4-[4-(benzyloxycarbonylamino)butyl]phenol (506 mg, 1.69mmol) and N-Boc bromide 19 (501 mg, 1.69 mmol) in anhydrous DMF (10 mL).The reaction mixture was stirred at 70° C. (oil bath) for 14 h, cooledto room temperature, and diluted with ethyl acetate (100 mL) and hexanes(20 mL). The mixture was washed with water (4×20 mL) and brine (40 mL)and concentrated under reduced pressure. Flash silica gel columnchromatography using ethyl acetate/CH₂Cl₂ (1:25, 1:20, v/v) gave thedesired product 20 as a thick yellow oil (718 mg, 83% yield). ¹H NMR(300 MHz, CDCl₃) δ 1.44 (s, 9H), 1.48-1.68 (m, 4H), 2.12-2.38 (m, 2H),2.48-2.60 (m, 2H), 3.10-3.24 (m, 2H), 3.75 (s, 3H), 3.97-4.06 (m, 2H),4.41-4.52 (m, 1H), 4.70 (br s, 1H), 5.09 (s, 2H), 5.25-5.37 (m, 1H),6.70-6.80 (m, 2H), 6.95-7.09 (m, 2H), 7.30-7.38 (m, 5H). m/z (ESI) 515[C₂₈H₃₈N₂O₇+H]⁺.

Example 93-{2-[4-(4-Benzyloxycarbonylaminobutyl)phenoxy]ethylamino}-2-N,N′-di-(tert-butoxycarbonyl)aminopropionicacid methyl ester (21)

3-{2-[4-(4-Benzyloxycarbonylaminobutyl)phenoxy]ethylamino}-2-N,N′-di-(tert-butoxycarbonyl)aminopropionicacid methyl ester (21)

A mixture of {4-[4-(2-aminoethoxy)phenyl]butyl}carbamic acid benzylester hydrochloride (141 mg, 0.372 mmol),2-[N,N′-di(tert-butoxycarbonyl)]aminoacrylic acid methyl ester (102 mg,0.338 mmol), and triethylamine (0.16 mL, 1.11 mmol) in methanol (3 mL)was stirred at 55° C. (oil bath) for 16 h. It was then cooled to roomtemperature. The solvent was removed by rotary evaporation. The residuewas taken up in ethyl acetate and washed with saturated sodiumbicarbonate solution and brine. The organic layer was concentrated invacuo and purified by flash silica gel column chromatography using ethylacetate/hexanes (gradient 1:10, 1:6, 1:4, and 1:2, v/v) to give thedesired Michael adduct 21 (110 mg, 51% yield). ¹H NMR (300 MHz, CDCl₃) δ1.42 (s, 9H), 1.43-1.50 (m, 9H), 1.50-1.65 (m, 4H), 2.50-2.60 (m, 2H),3.12-3.25 (m, 2H), 3.49-3.72 (m, 4H), 3.75 (s, 3H), 3.98-4.10 (m, 2H),4.45-4.65 (m, 1H), 4.79 (br s, 1H), 5.09 (s, 2H), 5.50 (m, 1H), 6.81 (d,J=8.5 Hz, 2H), 7.06 (d, J=8.5 Hz, 2H), 7.28-7.38 (m, 5H). m/z (ESI) 644[C₃₄H₄₉N₃O₉+H]⁺.

Example 105-[4-(4-Aminobutyl)phenyl]-2-(R)-tert-butoxycarbonylaminopentynoic acidmethyl ester (26)

[4-(4-Iodophenyl)but-3-ynyl]carbamic acid benzyl ester (22)

To a mixture of anhydrous THF and triethylamine (24 mL, 2/1, v/v) weresequentially added 1,4-diiodobenzene (2.03 g, 6.15 mmol) and copper (I)iodide (0.094 g, 0.246 mmol). The mixture was stirred at roomtemperature for 15 min. The flask was then evacuated and re-filled withArgon. The procedure was repeated three more times to ensure no oxygenremained. The catalyst, dichlorobis(triphenylphosphine)palladium(II)(0.173 g, 0.246 mmol) was added into the mixture under Argon protection.The other starting material, but-3-ynylcarbamic acid benzyl ester (0.50g, 2.46 mmol), dissolved in THF (8 mL) was added dropwise over 6 hours.The newly formed reaction mixture was further stirred at roomtemperature overnight. The solid in the reaction mixture was vacuumfiltered. The filtrate was concentrated. The residue was re-dissolved indichloromethane and purified by column chromatography, eluting with amixture of ethyl acetate (0-12%) and hexanes (100-88%) to afford theproduct 22 (0.852 g, 86%) as an off-white solid. ¹H NMR (300 MHz,CDCl₃): δ 2.61 (t, J=6.4 Hz, 2H), 3.44 (t, J=6.4 Hz, 2H), 5.07 (br s,1H), 5.12 (s, 2H), 7.10 (d, J=8.3 Hz, 2H), 7.35 (m, 5H), 7.63 (d, J=8.3Hz, 2H). m/z (APCI) 405 [C₁₈H₁₆NO₂+H]⁺.

2-(R)-tert-Butoxycarbonylaminopent-4-ynoic acid methyl ester (23)

The commercially available compound,2-(R)-tert-butoxycarbonylaminopent-4-ynoic acid (0.321 g, 1.50 mmol) wasdissolved in anhydrous DMF (5 mL). To the solution was added cesiumcarbonate (0.54 g, 1.65 mmol) in one portion. The mixture was stirred atroom temperature for 45 min before methyl iodide (0.20 mL, 3.00 mmol)was added, and further stirred for three hours. The reaction wasquenched with water (10 mL). The organics was extracted withdichloromethane (2×30 mL), washed with water (3×50 mL), and dried overanhydrous sodium sulfate. The solvent was completely removed undervacuum. The residue was further dried under high vacuum over night andused in the next reaction without further purification. The product 23was obtained as a colorless viscous oil (0.326 g, 95% yield). ¹H NMR(300 MHz, CDCl₃): δ 1.48 (s, 9H), 2.08 (t, J=2.6 Hz, 1H), 2.75 (t, J=6.3Hz, 2 H), 3.78 (s, 3H), 4.46 (m, 1H), 5.32 (br s, 5H).

5-[4-(4-Benzyloxycarbonylaminobut-1-ynyl)phenyl]-2-(R)-tert-butoxycarbonylaminopent-4-ynoicacid methyl ester (24)

Compound 22, [4-(4-iodophenyl)-but-3-ynyl]carbamic acid benzyl ester(0.52 g, 1.283 mmol) was dissolved in a mixture of anhydrous THF andtriethylamine (8 mL, 1/1, v/v). To the solution was added copper (I)iodide (0.025 g, 0.128 mmol). The mixture was stirred at roomtemperature for 15 min. The flask was then evacuated and re-filled withArgon. The procedure was repeated three more times to ensure no oxygenremained. The catalyst, dichlorobis(triphenylphosphine)palladium(II)(0.09 g, 0.128 mmol) was added into the mixture under Argon protection.The mixture was further stirred at room temperature for 30 min. Theother starting material 23, 2-tert-butoxycarbonylamino-pent-4-ynoic acidmethyl ester (0.32 g, 1.412 mmol), dissolved in THF (4 mL) was addeddropwise over 15 min. The newly formed reaction mixture was furtherstirred overnight at room temperature. The solid in the reaction mixturewas vacuum filtered. The filtrate was concentrated. The residue wasre-dissolved in dichloromethane and purified by column chromatography,eluting with a mixture of ethyl acetate (0-25%) and hexanes (100-75%) toafford the product 24 (0.64 g, 99%) as a gummy, yellowish solid. ¹H NMR(300 MHz, CDCl₃): δ 1.46 (s, 9H), 2.64 (t, J=6.3 Hz, 2H), 2.95 (t, J=4.1Hz, 2H), 3.42 (m, 2H), 3.79 (s, 3H), 4.56 (m, 1H), 5.12 (s, 2H), 5.38(br s, 1H), 7.29 (s, 4H), 7.35 (m, 5H). m/z (APCI) 502 [C₂₉H₃₂N₂O₆−H]⁺.

5-[4-(4-Aminobutyl)phenyl]-2-(R)-tert-butoxycarbonylaminopentynoic acidmethyl ester (26)

Compound 24,5-[4-(4-benzyloxycarbonylaminobut-1-ynyl)phenyl]-2-(R)-tert-butoxycarbonylaminopent-4-ynoicacid methyl ester (0.36 g, 0.713 mmol) was dissolved in a mixture ofethanol and methanol (50 mL, 1/1, v/v) and placed in a Parr shakerbottle. To the solution was added 10% Palladium on carbon (0.20 g, wet)in one portion under Argon protection. The flask was evacuated andre-filled with Argon. The procedure was repeated three more times. Themixture was then stirred at room temperature over night under 35 psihydrogen atmosphere. The flask was then evacuated and re-filled withnitrogen. The procedure was repeated three times. The catalyst was thenfiltered under vacuum and washed with ethanol (2×5 mL). The filtrate andwashings were combined and concentrated under reduced pressure. Theresidue was chromatographed over silica gel, eluting with a mixture ofmethanol (0-12%), ammonium hydroxide (0-1.2%) and di-chloromethane(100-86.8%), to afford two products, the desired product 26 (0.045 g,17%, a colorless, glass-like solid) and its protected form 25 (0.218 m,60%, a yellowish solid). ¹H NMR (300 MHz, CD₃OD) for compound 26: δ 1.43(s, 9H), 1.63-1.76 (m, 8H), 2.58-2.64 (m, 4H), 2.79 (t, J=7.0 Hz, 2H),3.69 (s, 3H), 4.10 (m, 1H), 7.09 (s, 4H). m/z (APCI) for compound 7: 379[C₁₂H₃₄N₂O₄+H]⁺.

¹H NMR (300 MHz, CD₃OD) for compound 25: δ 1.42 (s, 9H), 1.61-1.78 (m,8H), 2.54-2.68 (m, 4H), 2.92 (t, J=7.0 Hz, 2H), 3.14 (t, J=6.9 Hz, 2H),3.70 (s, 3H), 4.13 (m, 1H), 5.18 (s, 2H), 7.08 (s, 4H), 7.35 (m, 5H).

The compound 25 was resubmitted to hydrogenation to remove thebenzyloxycarbonyl protecting group. The procedure was performed asfollows: the compound 25 (0.218 g, 0.425 mmol) was dissolved in ethanol(10 mL). The solution was purged with nitrogen both before and after thepalladium catalyst (0.10 g, 10% on charcoal, 50% wet) was added, andsubjected to hydrogenation for two hours under atmospheric hydrogen. Thecatalyst was vacuum filtered and washed with ethanol (2×5 mL). Thefiltrate and washings were combined and concentrated under vacuum. Theresidue was chromatographed over silica gel, eluting with a mixture ofmethanol (0-14%), ammonium hydroxide (0-1.4%) and dichloromethane(100-84.6%), to afford the compound 26 (0.131 g, 81%).

Example 11 4-[4-(4-tert-Butoxycarbonylaminobutyl)phenylamino]butyricacid ethyl ester (27)

4-[4-(4-tert-Butoxycarbonylaminobutyl)phenylamino]butyric acid ethylester (27)

A solution of [4-(4-aminophenyl)butyl]carbamic acid tert-butyl ester(1.7 g, 6.43 mmol), 4-bromo-n-butyric acid ethyl ester (1.88 g, 9.64mmol), 4-methylmorpholine (1.0 mL, 9.64 mmol), and DMF (10 mL) wasstirred at 85° C. for 3 hours under a nitrogen atmosphere. The solventwas evaporated in vacuo. The residue was purified by flashchromatography (silica gel, 4:1, hexanes/ethyl acetate, v/v) to provide27 (0.44 g, 18%) as a colorless oil. ¹H NMR (300 MHz, CDCl₃) δ 1.23 (t,3H), 1.47 (s, 9H), 1.55 (m, 4H), 1.96 (m, 2H), 2.43 (t, 2H), 2.51 (t,2H), 3.19 (m, 4H), 4.14 (q, 2H), 6.54 (d, 2H), 7.00 (d, 2H). m/z (ESI)379.

Example 12 Synthesis of Amino Amides

Synthesis of {3-[4-(4-Aminobutyl)phenoxy]-1-carbamoylpropyl}carbamicacid tert-butyl ester (32) 2-Amino-4-hydroxybutyric acid methyl esterhydrochloride (28)

A suspension of DL-homoserine (1.00 g, 8.39 mmol) in methanol (40 mL)was placed in an ice bath. Trimethylsilyl chloride (2.34 mL, 18.5 mmol)was added dropwise via syringe. The reaction mixture gradually becamehomogenous and was further stirred at rt for 14 h, concentrated byrotary evaporation, and further dried under high vacuum. The crude oilthus obtained was used for the next step without further purification.¹H NMR (300 MHz, CD₃OD) δ 2.00-2.24 (m, 2H), 3.70-3.80 (m, 2H), 3.85 (s,3H), 4.12-4.22 (m, 1H). m/z (ESI) 134 [C₅H₁₁NO₃+H]⁺.

2-tert-Butoxycarbonylamino-4-hydroxybutyric acid methyl ester (29)

2-Amino-4-hydroxybutyric acid methyl ester hydrochloride (28) wassuspended in anhydrous THF (15 mL) and placed in an ice bath.Diisopropylethylamine (2.92 mL, 16.8 mmol) was added via syringe,followed by the addition of DMAP (205 mg, 1.68 mmol) and Boc₂O (3.85 g,17.6 mmol). The mixture was stirred at 0° C. for 10 min and at roomtemperature for 14 h. Solvent was removed under reduced pressure andresidue was taken up by ethyl acetate (100 mL), washed with water (30mL×2) and brine (40 mL), dried over sodium sulfate, and concentrated.The colorless oil (1.96 g) was used for the next step without furtherpurification. ¹H NMR (300 MHz, CDCl₃) δ 1.45 (s, 9H), 2.05-2.28 (m, 2H),3.68-3.75 (m, 2H), 3.80 (s, 3H), 4.08-4.15 (m, 1H), 5.30-5.41 (m, 1H).m/z (ESI) 234 [C₁₀H₁₉NO₅+H]⁺.

4-Bromo-2-tert-butoxycarbonylaminobutyric acid methyl ester (30)

A solution of triphenylphosphine (2.20 g, 8.39 mmol) in dry CH₂Cl₂ (20mL) was added dropwise via syringe to a solution of N-Boc homoserinemethyl ester 29 (8.39 mmol) and carbon tetrabromide (4.18 g, 12.60 mmol)in dry CH₂Cl₂ (20 mL). The resulting dark solution was stirred at roomtemperature for 16 h. Hexanes was added and precipitates were removed bysuction filtration. The filtrate was concentrated under reduced pressureand subject to flash silica gel column chromatography using ethylacetate/hexanes (1:10, v/v then 1:6, v/v) to give the desired product 30as a yellow oil (501 mg, 20% overall yield from homoserine). ¹H NMR (300MHz, CDCl₃) δ 1.45 (s, 9H), 2.12-2.48 (m, 2H), 3.39-3.47 (m, 2H), 3.78(s, 3H), 4.33-4.48 (m, 1H), 5.10-5.22 (m, 1H). m/z (ESI) 296[C₁₀H₁₈BrNO₄+H]⁺.

4-[4-(4-Benzyloxycarbonylaminobutyl)phenoxy]-2-tert-butoxycarbonylamino-butyricacid methyl ester (31)

Potassium carbonate (935 mg, 6.77 mmol) was added in one portion to asolution of 4-[4-(benzyloxycarbonylamino)butyl]phenol (506 mg, 1.69mmol) and N-Boc bromide 30 (501 mg, 1.69 mmol) in anhydrous DMF (10 mL).The reaction mixture was stirred at 70° C. (oil bath) for 14 h, cooledto room temperature, and diluted with ethyl acetate (100 mL) and hexanes(20 mL). The mixture was washed with water (4×20 mL ) and brine (40 mL)and concentrated under reduced pressure. Flash silica gel columnchromatography using ethyl acetate/CH₂Cl₂ (1:25, 1:20, v/v) gave thedesired product 31 as a thick yellow oil (718 mg, 83% yield). ¹H NMR(300 MHz, CDCl₃) δ 1.44 (s, 9H), 1.48-1.68 (m, 4H), 2.12-2.38 (m, 2H),2.48-2.60 (m, 2H), 3.10-3.24 (m, 2H), 3.75 (s, 3H), 3.97-4.06 (m, 2H),4.41-4.52 (m, 1H), 4.70 (br s, 1H), 5.09 (s, 2H), 5.25-5.37 (m, 1H),6.70-6.80 (m, 2H), 6.95-7.09 (m, 2H), 7.30-7.38 (m, 5H). m/z (ESI) 515[C₂₈H₃₈N₂O₇+H]⁺.

{4-[4-(3-tert-Butoxycarbonylamino-3-carbamoylpropoxy)phenyl]butyl}carbamicacid benzyl ester (32)

Ammonia (7 M in methanol, 20 mL) was added to a solution of N-Boc methylester 31(718 mg, 1.40 mmol) in methanol (5 mL) and the mixture wasstirred at room temperature in a sealed tube for 40 h. The mixture wasconcentrated by rotary evaporation and purified by flash silica gelcolumn chromatography using methanol/dichloromethane (1:30, 1:20, v/v)to give the desired amide 32 as a white solid (436 mg, 63% yield). ¹HNMR (300 MHz, CDCl₃) δ 1.45 (s, 9H), 1.48-1.68 (m, 4H), 2.09-2.32 (m,2H), 2.48-2.61 (m, 2H), 3.08-3.25 (m, 2H), 3.97-4.20 (m, 2H), 4.30-4.45(m, 1H), 4.75 (br s, 1H), 5.09 (s, 2H), 5.48-5.58 (m, 1H), 5.62 (br s,1H), 6.38 (br s, 1H), 6.75-6.85 (m, 2H), 6.99-7.10 (m, 2H), 7.28-7.40(m, 5H). m/z (ESI) 500 [C₂₇H₃₇N₃O₆+H]⁺.

Example 13 [4-(4-Benzyloxycarbonylaminobutyl)phenoxy]acetic acid ethylester (33)

Synthesis of [4-(4-Benzyloxycarbonylaminobutyl)phenoxy]acetic acid ethylester (33)

A solution of [4-(4-hydroxyphenyl)butyl]carbamic acid benzyl ester (2.0g, 6.7 mmol), potassium carbonate (1.0 g, 7.3 mmol), sodium iodide (0.4g, 2.7 mmol), and DMF (10 mL) was stirred for 30 minutes. A solution ofethyl bromoacetate (0.8 mL, 7.4 mmol) in DMF (10 mL) was added dropwiseto the reaction. The reaction was further stirred at room temperaturefor 3 days, and then poured into water (200 mL). The product wasextracted with ethyl acetate. The organic layer was dried over sodiumsulfate and concentrated in vacuo. The residue was purified by flashchromatography (silica gel, 1:5 ethyl acetate/hexanes, v/v) to providethe desired product 33 (2.3 g, 89%) as a white solid. ¹H NMR (300 MHz,CDCl₃) δ 1.34 (t, 3H), 1.57 (m, 4H), 2.56 (t, 2H), 3.20 (q, 2H), 4.25(q, 2H), 4.59 (s, 2H), 5.10 (s, 2H), 6.85 (d, 2H), 7.06 (d, 2H), 7.38(m, 5H).

Example 14 Synthesis of Thia Amides

N-{4-[4-(Dimethylthiocarbamoyloxy)phenyl]butyl}phthalimide (34)

A suspension of sodium hydride in mineral oil (0.44 g of 60%) inanhydrous DMF (10 mL) was cooled to 0° C.N-[4-(4-hydroxyphenyl)butyl]phthalimide (2.95 g, 10 mmol) dissolved indry DMF (15 ml) was added into the mixture which was then stirred for 30min at 0° C. and allowed to warm to room temperature over 1 h. To themixture was added portionwise N,N-dimethylthiocarbamoyl chloride (1.35g, 11 mmol) dissolved in DMF (10 ml). The newly formed mixture wasstirred at room temperature overnight, then at 50° C. for 1 h, and wascooled back to room temperature, at which point methanol (10 mL) wasadded into the mixture. The solvent was removed under reduced pressure.The residue was purified by flash chromatography over silica gel(dichloromethane/hexane/ethyl acetate, 10:1:0.2, v/v) to giveN-{4-[4-(dimethythiocarbamoyloxy)phenyl]butyl}phthalimide 34 (2.27 g,59%) as a slightly yellow solid. ¹H NMR (300 MHz, CDCl₃) δ 1.72 (m, 4H),2.66 (m, 2H), 3.33 (s, 3H), 3.45 (s, 3H), 3.70 (m, 2H), 6.96 (d, 2H),7.18 (d, 2H), 7.71 (m, 2H), 7.84 (m, 2H). m/z (ESI) 383[C₂₁H₂₂N₂O₃S+H]⁺.

N-{4-[4-(Dimethylcarbamoylthio)phenyl]butyl}phthalimide (35)N-{4{4-[(Dimethythiocarbamoyloxy)phenyl]butyl}phthalimide 34

(2.1 g, 5.4 mmol) was placed in preheated sand bath at 230° C. Thetemperature was raised to 280° C. and the melted compound was kept atthis temperature for 2 h in argon atmosphere. The mixture was cooled andthe residue was purified by flash chromatography over silica gel(dichloromethane/hexane/ethyl acetate,10:1:0.2, v/v) to give 35 (1.2 g,57%) as a white powder. ¹H NMR (300 MHz, CDCl₃) δ 1.70 (m, 4H), 2.66 (t,2H), 3.05 (br s, 3H), 3.71 (t, 2H), 6.96 (d, 2H), 7.19 (d, 2H), 7.38 (d,2H), 7.70 (m, 2H), 7.83 (m, 2H). m/z (ESI) 383 [C₂₁H₂₂N₂O₃S+H]⁺.

{4-[4-(Dimethylcarbamoylthio)phenyl]butylamine (36)

Phthalimide 35 (1.1 g, 2.8 mmol) was dissolved in a solution containing6.6% methylamine in ethanol (100 mL) and allowed to stir at roomtemperature overnight. The solvent was removed under reduced pressureand the residue was purified by flash chromatotography (silica gel,chloroform/methanol/ammonium hydroxide, 10:1:0.1, v/v) to afford thefree amine 36 (0.31 g, 42%) as a white powder. ¹H NMR (300 MHz, CD₃OD) δ1.51 (m, 2H), 1.65 (m, 2H), 2.66 (m, 4H), 3.02 (m, 6H), 7.23 (d, 2H),7.35 (d, 2H).

N-tert-Butoxycarbonyl-{4-[4-(dimethylcarbamoylthio)phenyl]butylamine(37)

Di-tert-butyldicarbonate (0.35 g, 1.6 mmol) was added to a solution of36 (0.31 g, 1.22 mmol), 4-Dimethylaminopyridine (DMAP), anddichloromethane (50 mL). The mixture was stirred at room temperature for26 h. The solvent was removed under reduced pressure and the residue waspurified by flash chromatography over silica gel (hexane/ethyl acetate,3:1, v/v) to give 36 (0.36 g, 83%) as a white powder. ¹H NMR (300 MHz,CDCl₃) δ 1.44 (s, 9H), 1.51 (m, 2H) 1.64 (m, 2H), 3.08 (m, 8H), 4.48 (brs, 1H), 7.18 (d, 2H), 7.39 (d, 2H).

N-[4-(4-Mercaptophenyl)butyl]carbamic acid tert-butyl ester (38)

N-tert-Butoxycarbonyl-{4-[4-(dimethylcarbamoylthio)phenyl]butylamine 37(0.35 g, 1.02 mmol) was dissolved in MeOH (8 mL). KOH (0.19 g, 3.4 mmol)dissolved in water (2 ml) was added. The mixture was stirred underreflux for 6 h and cooled to room temperature. The solvent was removedunder reduced pressure. The residue was dissolved in water and acidifiedwith 5% aqueous HCl to pH ˜5. The solvent was removed again underreduced pressure and the residue was purified by flash chromatographyover silica gel (hexane/ethyl acetate, 3:1, v/v) to give the desiredthiophenol 38 (0.13 g, 45%) as a clear oil. ¹H NMR (300 MHz, CDCl₃) δ1.43 (s, 9H), 1.49 (m, 2H) 1.59 (m, 2H), 2.57 (t, 2H), 3.12 (m, 2H),3.38 (s, 1H), 4.47 (br s, 1H), 7.04 (d, 2H), 7.19 (d, 2H).

Example 15 4-(4-Carboxymethylphenyl)butylamine (41) Methanesulfonic acid4-(4-carboxymethylphenyl)butyl ester (39)

Compound 39 was prepared according to the published procedure¹. ¹H NMR(300 MHz, CDCl₃) δ 1.75 (m, 4H), 2.78 (m, 2H), 3.12 (s, 3H), 3.88 (s,3H), 4.22 (m, 2H), 7.28 (d, 2H), 7.98 (d, 2H)

4-(4-Carboxymethylphenyl)butylazide (7). Typical procedure C

Compound 39 (6 g, 0.02 mol) was dissolved in 80 ml of dry DMF thensodium azide (1.8 g, 0.027 mol) was added. The suspension was stirred at80° C. (oil bath) for 3 h. The solvent was then removed at reducedpressure and the residual oil was treated with CH₂Cl₂ (100 mL). Theresulting solution was washed with water (2×100 mL), brine and driedover magnesium sulfate. The solvent was removed under reduced pressurethen the residue was redissolved in a 1:1 mixture of ethylacetate/hexanes (200 mL) and passed through a pad of silica gel. Thesolvent was removed under reduced pressure to give 4.1 g (85%) of 7 asclear oil. ¹H NMR (300 MHz, CDCl₃) δ 1.68 (m, 4H), 2.22 (t, 2H), 3.29(t, 3H), 3.92 (s, 3H), 7.28 (d, 2H), 7.98 (d, 2H).

4-(4-Carboxymethylphenyl)butylamine (41)

Azide 40 (1.7 g, 7.2 mmol) and triphenylphosphine (1.9 g, 7.2 mmol) weredissolved in a 10% solution of water in THF (66 mL) and stirredovernight at 25° C. Then more triphenylphosphine (0.8 g, 3 mmol) wasadded and the heating was continued at 60° C. (oil bath) for 6 h. Thesolvent was removed under reduced pressure and the residue was treatedwith 2M HCl (100 mL) and extracted with ethyl acetate (2×50 mL). Thewater fraction was collected and ammonium hydroxide was added until thepH reached approximately 13. The mixture was extracted with ethylacetate (2×100 mL) then the organic fraction was washed with brine,water and dried with sodium sulfate. Ethyl acetate was removed underreduced pressure to give 0.8 g (53%) of amine 41.

Example 16 Synthesis of [4-(4-Aminobutyl)phenoxy]acetic acid ethyl ester(43) [4-(4-Benzyloxycarbonylaminobutyl)phenoxy]acetic acid ethyl ester(42)

Sodium hydride (60% dispersion in mineral oil) (0.24 g, 10.05 mmol) wasadded to a cold (0° C.) solution of 4-(4-hydroxyphenyl)butylamine (2 g,6.68 mmol) in THF (150 mL) under nitrogen atmosphere. The reactionmixture was allowed to warm up to room temperature over 0.5 h withstirring, then ethyl bromoacetate (0.96 mL, 8.02 mmol) andtetrabutylammonium iodide (0.25 g, 0.67 mmol) was sequentially added.The reaction was further stirred at room temperature overnight. Silicagel (25 mL) was added into the mixture and the solvent was evaporated.The impregnated silica gel was subjected to column chromatographypurification (silica gel, 5:1 hexanes/ethyl acetate). 2.42 g (94%) of 42was obtained as a white solid. ¹H NMR (300 MHz, CDCl₃) δ 1.30 (t, 3H),1.57 (m, 4H), 2.58 (m, 2H), 3.20 (m, 2H), 4.28 (m, 2H), 4.58 (s, 2H),4.74 (br s, 1H), 5.10 (br s, 2H), 6.82 (m, 2H), 7.08 (m, 2H), 7.38 (brs, 5H).

[4-(4-Aminobutyl)phenoxy]acetic acid ethyl ester (43)

A suspension of 42 (1.11 g, 2.88 mmol) and 10% palladium on carbon (0.40g, wet) in methanol (50 mL) was stirred at room temperature for 2 hunder atmospheric pressure of hydrogen. The mixture was then filteredthrough a silica gel pad. The solvent was evaporated to provide 43(0.64g, 88%) as a white solid. ¹H NMR (300 MHz, DMSO-d₆) δ 1.21 (m, 3H),1.30-1.63 (m, 6H), 3.13 (m, 2H), 4.17 (m, 2H), 4.72 (br s, 2H), 6.84 (m,2H), 7.10 (m, 2H).

Example 17 Synthesis of(4-{4-[N′-(3,5-diamino-6-chloropyrazine-2-carbonyl)guanidino]butyl}-phenoxy)aceticacid 2-aminoethyl ester dihydrochloride (PSA 25454)

[4-(4-Benzyloxycarbonylaminobutyl)phenoxy]acetic acid2-tert-butoxycarbonylamino-ethyl ester (44a)

[4-(4-Benzyloxycarbonylaminobutyl)phenoxy]acetic acid 48 (240 mg, 0.67mmol) was dissolved in anhydrous CH₂Cl₂ (6 mL). To the solution wasadded DMAP (98 mg, 0.80 mmol), followed by EDC.HCl (154 mg, 0.81 mmol).The reaction mixture was stirred at room temperature for 15 min.(2-Hydroxyethyl)carbamic acid tert-butyl ester (0.21 mL, 1.34 mmol) wasthen added and the stirring was continued for 16 h. Solvent was removedby rotary evaporation. The residue was subjected to flash silica gelcolumn chromatography eluting with ethyl acetate/dichloromethane (1:20,1:15, and 1:10, v/v) to give the desired ester 44a as a colorless oil(162 mg, 48% yield). ¹H NMR (300 MHz, CDCl₃) δ 1.35-1.65 (m, 4H), 1.44(s, 9H), 2.45-2.58 (m, 2H), 3.10-3.22 (m, 2H), 3.25-3.42 (m, 2H),4.18-4.28 (m, 2H), 4.62 (s, 2H), 4.70 (br s, 1H), 4.84 (br s, 1H), 5.08(s, 2H), 6.81 (d, 2H), 7.07 (d, 2H), 7.25-7.38 (m, 5H). m/z (ESI) 501[C₂₇H₃₆N₂O₇+H]⁺.

[4-(4-Aminobutyl)phenoxy]acetic acid 2-tert-butoxycarbonylaminoethylester ethyl acetate (45a)

A solution of [4-(4-benzyloxycarbonylaminobutyl)phenoxy]acetic acid2-tert-butoxycarbonylaminoethyl ester 44a (162 mg, 0.32 mmol) and aceticacid (3 drops) in ethyl acetate (3 mL) and dichloromethane (6 mL) wasstirred at room temperature for 16 h under hydrogen atmosphere in thepresence of palladium hydroxide on activated charcoal (80 mg, 60% wet).The catalyst was removed by suction filtration and the liquid filtratewas concentrated in vacuo to give [4-(4-aminobutyl)phenoxy]acetic acid2-tert-butoxycarbonylaminoethyl ester ethyl acetate 45a as an off-whitesolid (100 mg, 84% yield) which was used directly without purification.m/z (ESI) 367 [C₁₉H₃₀N₂O₅+H]⁺.

(4-{4-[N′-(3,5-Diamino-6-chloropyrazine-2-carbonyl)guanidino]butyl}phenoxy)aceticacid 2-tert-butoxycarbonylaminoethyl ester (46a, PSA 25309)

A solution of [4-(4-aminobutyl)phenoxy]acetic acid2-tert-butoxycarbonylaminoethyl ester ethyl acetate 45a (50 mg, 0.27mmol) and triethylamine (0.27 mL, 1.53 mmol) in anhydrous THF (4 mL) wasstirred at 55° C. (oil bath) for 15 min. To the solution was added1-(3,5-diamino-6-chloropyrazine-2-carbonyl)-2-methylisothioureahydriodide (130 mg, 0.34 mmol) in two portions during a period of 15min. The reaction mixture was stirred at 55° C. for 1 h and cooled toroom temperature. The mixture was concentrated by rotary evaporation.The crude residue was purified by flash silica gel column chromatographyeluting with dichloromethane/methanol/ concentrated ammonium hydroxide(200:10:0, 200:10:1, and 150:10:1, v/v) to give(4-{4-[N′-(3,5-diamino-6-chloropyrazine-2-carbonyl)guanidino]butyl}phenoxy)aceticacid 2-tert-butoxycarbonylaminoethyl ester 46a (PSA 25309) as a yellowsolid (60 mg, 76% yield). An analytical pure sample was obtained bysemi-preparative HPLC purification (acetonitrile/water). mp 80-82° C. ¹HNMR (300 MHz, CD₃OD) δ 1.45 (s, 9H), 1.60-1.78 (m, 4H), 2.56-2.66 (m,2H), 3.30-3.38 (m, 4H), 4.15-4.21 (m, 2H), 4.64 (s, 2H), 6.81 (d, 2H),7.10 (d, 2H). m/z (ESI) 579 [C₂₅H₃₅ClN₈O₆+H]⁺.

(4-{4-[N-(3,5-Diamino-6-chloropyrazine-2-carbonyl)guanidino]butyl}phenoxy)aceticacid 2-aminoethyl ester dihydrochloride (47a, PSA 25454)

(4-{4-[N′-(3,5-Diamino-6-chloropyrazine-2-carbonyl)guanidino]butyl}phenoxy)aceticacid 2-tert-butoxycarbonylaminoethyl ester 46a (24 mg, 0.041 mmol) wastreated with HCl (4 N in dioxane, 1 mL, 4 mmol) at room temperature for14 h. The reaction mixture was concentrated in vacuo and the residue waspurified by semi-preparative HPLC method (acetonitrile/water) to affordthe desired product 47a (PSA 25454, 7 mg, 31%) as a yellow solid. mp66-68° C. ¹H NMR (500 MHz, CD₃OD) δ 1.65-1.76 (m, 4H), 2.60-2.68 (m,2H), 3.25-3.31 (m, 2H), 3.33-3.39 (m, 2H), 4.40-4.45 (m, 2H), 4.75 (s,2H), 6.86 (d, 2H), 7.11 (d, 2H). m/z (APCI) 479 [C₂₀H₂₇ClN₈O₄+H]⁺.

Example 18 Synthesis of(4-{4-N′-(3,5-diamino-6-chloropyrazine-2-carbonyl)guanidino]butyl}-phenoxy)aceticacid 2-piperidin-1-yl-ethyl ester (PSA 25453)

[4-(4-Benzyloxycarbonylaminobutyl)phenoxy]acetic acid2-piperidin-1-yl-ethyl ester (44b)

Following the same procedure for the synthesis of compound 44a, compound49b was synthesized from compound 48 in 60% yield as a colorless oil. ¹HNMR (300 MHz, CDCl₃) δ 1.33-1.68 (m, 10H), 2.30-2.45 (m, 4H), 2.50-2.65(m, 4H), 3.10-3.25 (m, 2H), 4.28-4.35 (m, 2H), 4.60 (s, 2H), 4.75 (br s,1H), 5.08 (s, 2H), 6.80 (d, 2H), 7.05 (d, 2H), 7.28-7.38 (m, 5H). m/z(ESI) 469 [C₂₇H₃₆N₂O₅+H]⁺.

[4-(4-Aminobutyl)phenoxy]acetic acid 2-piperidin-1-yl-ethyl ester ethylacetate (45b)

Following the same procedure for the synthesis of compound 45a, compound45b was synthesized from compound 44b in 80% yield as an off-whitesolid, and used directly without purification. m/z (ESI) 335[C₁₉H₃₀N₂O₃+H]⁺.

(4-{4-[N-(3,5-Diamino-6-chloropyrazine-2-carbonyl)guanidino]butyl}phenoxy)aceticacid 2-piperidin-1-yl-ethyl ester (46b, PSA 25453)

Following the same procedure for the synthesis of compound 46a, compound46b was synthesized from compound 45b. One fifth of the crude reactionmixture, after concentration under vacuum, was purified bysemi-preparative HPLC method (acetonitrile/water) to afford pure 46b(PSA 25453) as a yellow solid. mp 60-62° C. ¹H NMR (500 MHz, DMSO-d₆) δ1.30-1.80 (m, 10H), 2.50-2.60 (m, 2H), 2.88-2.98 (m, 2H), 3.25-3.35 (m,2H), 3.35-3.48 (m, 4H), 4.42-4.48 (m, 2H), 4.78 (s, 2H), 6.88 (d, 2H),7.12 (d, 2H), 7.42 (s, 2H), 8.75 (s, 1H), 8.90 (s, 1H), 9.15 (s, 1H),9.65 (s, 1H), 10.45 (s, 1H). m/z (ESI) 547 [C₂₅H₃₅ClN₈O₄+H]⁺.

Example 19 Synthesis of(4-{4-[N′-(3,5-diamino-6-chloropyrazine-2-carbonyl)guanidino]butyl}-phenoxy)aceticacid piperidin-4-yl-methyl ester dihydrochloride (PSA 25720)

4-{2-[4-(4-Benzyloxycarbonylaminobutyl)phenoxy]acetoxymethyl}piperidine-1-carboxylicacid tert-butyl ester (44c)

Following the same procedure for the synthesis of compound 44a, compound44c (a colorless oil) was synthesized in 71% yield by the reaction ofcompound 48 with 4-hydroxymethylpiperidine-1-carboxylic acid tert-butylester, synthesis of which is described below. ¹H NMR (300 MHz, CDCl₃) δ1.05-1.20 (m, 2H), 1.45 (s, 9H), 1.50-1.65 (m, 6H), 1.70-1.88 (m, 1H),2.50-2.75 (m, 4H), 3.12-3.26 (m, 2H), 4.00-4.15 (m, 4H), 4.60 (s, 2H),4.88 (br s, 1H), 5.08 (s, 2H), 6.80 (d, 2H), 7.08 (d, 2H), 7.30-7.48 (m,5H). m/z (ESI) 555 [C₃₁H₄₂N₂O₇+H]⁺.

Synthesis of 4-hydroxymethylpiperidine-1-carboxylic acid tert-butylester

To a solution of piperidin-4-yl-methanol (2.59 g, 22.5 mmol) in1,4-dioxane (60 mL) was added aqueous sodium hydroxide (1 N, 35 mL). Themixture was chilled in an ice bath. To the chilled mixture was addedBoc₂O (7.36 g, 33.7 mmol) in one portion. The reaction mixture wasstirred at 0° C. for 30 min and at room temperature for 16 h, andconcentrated under vacuum to about one-third of its original volume. Theremaining mixture was diluted with water and dichloromethane. Layerswere separated and the aqueous layer was further extracted withdichloromethane. The combined organics were concentrated under vacuumand purified by flash silica gel column chromatography eluting withethyl acetate/hexanes (1:10, 1:4, and 1:1, v/v) to give4-hydroxymethylpiperidine-1-carboxylic acid tert-butyl ester (4.71 g,97% yield) as a white solid. ¹H NMR (300 MHz, CDCl₃) δ 1.10-1.20 (m,2H), 1.30 (t, 1H), 1.47 (s, 9H), 1.60-1.69 (m, 1H), 1.69-1.75 (m, 2H),2.65-2.75 (m, 2H), 3.48-3.52 (m, 2H), 4.05-4.18 (m, 2H). m/z (ESI) 216[C₁₁H₂₁NO₃+H]⁺.

4-{2-[4-(4-Aminobutyl)phenoxy]acetoxymethyl}piperidine-1-carboxylic acidtert-butyl ester ethyl acetate (45c)

Following the same procedure for the synthesis of compound 45a, compound45c was synthesized from compound 44c in 99% yield as an off-whitesolid, and used directly without purification. m/z (ESI) 421[C₂₃H₃₆N₂O₅+H]⁺.

4-[2-(4-{4-N′-(3,5-Diamino-6-chloropyrazine-2-carbonyl)guanidino]butyl}phenoxy)-acetoxymethyl]piperidine-1-carboxylicacid tert-butyl ester (46c)

Following the same procedure for the synthesis of compound 46a, compound46c was synthesized from compound 45c. One-third of the crude reactionmixture, after concentration under vacuum, was purified bysemi-preparative HPLC method (acetonitrile/water) to give 46c as ayellow solid. m/z (ESI) 633 [C₂₉H₄₁ClN₈O₆+H]⁺.

(4-{4-[N′-(3,5-Diamino-6-chloropyrazine-2-carbonyl)guanidino]butyl}phenoxy)aceticacid piperidin-4-yl-methyl ester dihydrochloride (47c, PSA 25720)

Following the same procedure for the synthesis of compound 47a, compound47c (PSA 25720) was synthesized from compound 46c in 39% yield as ayellow solid. mp 100-102° C. ¹H NMR (500 MHz, DMSO-d₆) δ 1.30-1.45 (m,2H), 1.50-1.65 (m, 4H), 1.72-1.80 (m, 2H), 1.88-1.98 (m, 1H), 2.52-2.60(m, 2H), 2.78-2.88 (m, 2H), 3.20-3.29 (m, 2H), 4.00 (d, 2H), 4.75 (s,2H), 6.85 (d, 2H), 7.12 (d, 2H), 7.40 (br s, 2H), 8.80 (br s, 3H), 9.20(br s, 2H). m/z (APCI) 533 [C₂₄H₃₃ClN₈O₄+H]⁺.

Example 20 Sodium Channel Blocking Activity of Selected Soluble Esters

Utilizing the tests set forth above, Table 4 summaries the ENaC blockingability of some of the Esters of this invention when assayed in caninebronchial epithelium.

TABLE 4 Epithelial Sodium Channel Blocking Activity of Selected Esters

IC₅₀ PSA# (nM) Fold Amiloride** (PSA4022 = 100) 25309 4 119 25453 19 ±16 38 (n = 5) 25454 12 ± 2  44 (n = 6) 25720 3 ± 0 189 (n = 4)**Relative potency for PSA4022 = 100 using IC₅₀ from PSA4022 in samerun.

Obviously, numerous modifications and variations of the presentinvention are possible in light of the above teachings. It is thereforeto be understood that within the scope of the appended claims, theinvention may be practiced otherwise than as specifically describedherein.

-   References. 1. Rappoport, D. A.; Hassid, Z.; J. Amer. Chem. Soc.,    1951, 73, 5524-5525, incorporated herein by reference.

1-154. (canceled)
 155. A pyrazinoylguanidine compound represented by theformula:

or a pharmaceutically acceptable salt thereof.
 156. The compound ofclaim 155, which is a pharmaceutically acceptable salt.
 157. Thecompound of claim 155, which is an acid addition salt of an inorganicacid or an organic acid.
 158. The compound of claim 155, which is anacid addition salt of methanesulfonic acid.
 159. A pharmaceuticalcomposition, comprising the compound of claim 155 and a pharmaceuticallyacceptable carrier.
 160. A method of administering the compound of claim155 to a mucosal surface, comprising administering the compound to amusosal surface of a subject.
 161. The method of claim 160, wherein thecompound is administered topically, orally, rectally, vaginally,ocularly and/or dermally.
 162. A method of administering the compound ofclaim 155 to an airway surface, comprising administering the compound toan airway surface of a subject.
 163. The method of claim 162, whereinthe compound is administered in the form of a spray, mist or droplet.164. The method of claim 163, wherein the spray, mist or dropletcontains physiological or dilute saline.
 165. The method of claim 162,wherein the compound is administered in the form of respirableparticles.
 166. The method of claim 165, wherein the respirableparticles have an average size of about 1 to 5 microns.
 167. The methodof claim 162, wherein the compound is in the form of an aerosolsuspension of respirable particles.
 168. The method of claim 167,wherein the respirable particles have an average size of about 1 to 5microns.
 169. The method of claim 162, wherein the compound isadministered to the lungs of the subject using an inhaler.
 170. Themethod of claim 169, wherein the aerosol is produced by an aerosolgenerator.
 171. The method of claim 170, wherein the aerosol generatoris a metered dose inhaler.
 172. The method of claim 170, wherein theaerosol generator is an insufflator.
 173. The method of claim 170,wherein the aerosol generator is a nebulizer.
 174. The method of claim167, wherein the aerosol contains solid particles.
 175. The method ofclaim 174, wherein the particles are produced by an aerosol generator ata rate of about 10 to 150 liters per minute.
 176. The method of claim167, wherein the aerosol contains liquid particles.
 177. The method ofclaim 176, wherein the particles are produced by an aerosol generator ata rate of about 10 to 150 liters per minute.
 178. The method of claim176, wherein the aerosol is produced by a pressure driven aerosolnebulizer or ultrasonic nebulizer.